T1e-SENSITIVE INVERSION-RECOVERY IMAGING FOR EPROI

ABSTRACT

An apparatus and method for improved S/N measurements useful for electron paramagnetic resonance imaging in situ and in vivo, using high-isolation transmit/receive surface coils and temporally spaced pulses of RF energy (e.g., in some embodiments, a RF pi pulse) having an amplitude sufficient to rotate the magnetization by 180 degrees followed after varied delays, by a second RF pulse having an amplitude half that of the initial pulse to rotate the magnetization by, e.g., 90 degrees (a pi/2 pulse), to the plane orthogonal to the static field where it evolves for a short time. Then a third RF pi pulse sufficient to rotate the magnetization by, e.g., 180 degrees, forms an echo (in some embodiments, the second and third pulses are from the same signal as the first pulse but are phase shifted by 0, 90, 180, or 270 degrees to reduce signal artifact), to image human body.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/213,768 titled “Method and apparatus for T_(1e)-sensitiveinversion-recovery imaging for EPRI,” filed Jul. 19, 2016 by Howard J.Halpern et al. (which will issue as U.S. Pat. No. 11,612,336 on Mar. 28,2023), which is a divisional of U.S. patent application Ser. No.13/032,637 titled “T₁-sensitive inversion-recovery-imaging method andapparatus for EPRI,” filed Feb. 22, 2011 by Howard J. Halpern et al.(which issued as U.S. Pat. No. 9,392,957 on Jul. 19, 2016), which claimspriority to U.S. Provisional Patent Application 61/306,917 titled“High-isolation transmit/receive surface coils and method for EPRI”filed Feb. 22, 2010 by Howard J. Halpern, U.S. Provisional PatentApplication 61/356,555 titled “T₁-sensitive inversion recovery imagingapparatus and method for EPRI” filed Jun. 18, 2010 by Howard J. Halpernet al., and U.S. Provisional Patent Application 61/445,037 titled“T₁-sensitive inversion recovery imaging method and apparatus for EPRI”filed Feb. 21, 2011 by Howard J. Halpern et al., which are allincorporated herein by reference in their entirety including theirappendices.

This application is related to U.S. patent application Ser. No.13/032,626 titled “High-isolation transmit/receive surface coils andmethod for EPRI” filed Feb. 22, 2011 by Howard J. Halpern (which issuedas U.S. Pat. No. 8,664,955 on Mar. 4, 2014), which is incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under EB002034, R01CA098575 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to the field of medical imaging and modeling, andmore specifically to a method and apparatus for improved signal-to-noise(S/N) measurements useful for electron paramagnetic resonance imaging(EPRI), in situ and in vivo, using a series of transmitted pulse sets ofvariously temporally spaced-apart and phase-shifted pulses oftransmitted radio frequency (RF) energy that disturb the electron-spinalignment, and then receive the much-weaker resulting RF signal as theelectron spins realign to the static magnetic field. The transmittedsignal is used in a set of static magnetic-field coils (which generatethe electron-spin-aligning constant and gradient magnetic fields) andhigh-isolation transmit/receive surface, volume, or surface-volume coilsthat are configured to reduce the reception of the transmitted RF pulsesby the receive coils. The received signal is then quadrature decoded (todigitized I values and Q values) using a reference RF signal. In someembodiments, a triplet set of pulses (e.g., in some embodiments, eachmeasurement uses a triplet pulse set of three RF pulses having aselected pulse-to-pulse-to-pulse temporal spacing, a selected set ofpulse amplitudes, and various selected phase-shift amounts relative to areference RF signal, for example:

(1) a first RF “pi” pulse (e.g., about 35 ns of RF (=about 9 cycles of asignal that is about 250 MHz that cause the electron spins to rotate by180 degrees (pi radians) relative to their orientation in the referenceRF signal) of a given “full magnitude” followed, after a firstpredetermined time delay by(2) a second pulse of about 9 cycles of substantially the same frequencybut having half the magnitude (one-quarter the power) and a shiftedphase (by one of four different amounts: about 0 degrees, about 90degrees, about 180 degrees, or about 270 degrees), relative to thecycles of the first pulse (in some embodiments, the cycles of the secondpulse are obtained from the same reference RF signal source as those ofthe first pulse but are phase shifted by the selected amount for thatset of three pulses), (the second pulse causes the directions of theelectron spins to rotate 90 degrees (pi/2 radians)) and then followed,after a second predetermined time delay, by(3) a third pulse of about nine (9) cycles of substantially the samefrequency but having a full magnitude (the same magnitude as the firstpulse) and another shifted phase (by one of four different amounts:about 0 degrees, about 90 degrees, about 180 degrees, or about 270degrees), relative to the cycles of the first pulse (in someembodiments, the approximately nine cycles of the third pulse are alsoobtained from the same signal source as those of the first pulse but arephase shifted by the selected amount and have the same full magnitude).The second pulse causes the directions of the electron spins to rotate180 degrees (pi radians), which causes a spin echo of the T₁ relaxationsignal. The T₁ spin echo is temporally separated from the thirdexcitation pulse enough to allow better sensing (better signal-to-noiseratio (SNR)). In some embodiments, a plurality of series of pulses areused, wherein each series has sixteen (16) triplet sets of pulses,wherein each triplet set uses one of four different phase-shift amountsfor the second pulse and one of four different phase-shift amounts forthe third pulse, and each series uses one of a plurality ofpredetermined first time delays and one of a plurality of predeterminedsecond time delays. By using pulses that sense the T₁ signal rather thanthe T₂ signal, the present invention provides, in some embodiments,improved micro-environmental images that are representative ofparticular internal structures in the human body and spatially resolvedimages of tissue/cell protein signals responding to conditions (such ashypoxia) that show the temporal sequence of certain biologicalprocesses, and, in some embodiments, that distinguish malignant tissuefrom healthy tissue.

BACKGROUND OF THE INVENTION

Cells activate protein signaling in response to crucial environmentalconditions. Among the best studied is the cellular response tochronically low levels of oxygen, hypoxia. Cells respond to hypoxia byincreasing hypoxia inducible factor 1α (HIF1α), a signaling peptidewhich is the master regulator of hypoxic response. HIF1α promotes genesand their protein products, orchestrating cell, tissue, and organismhypoxic response such as new vessel formation and increase in red cellvolume.

U.S. Pat. No. 6,977,502 to David Hertz issued Dec. 20, 2005 titled“Configurable matrix receiver for MRI” is incorporated herein byreference. Hertz describes a configurable matrix receiver having aplurality of antennas that detect one or more signals. The antennas arecoupled to a configurable matrix comprising a plurality of amplifiers,one or more switches that selectively couple the amplifiers in seriesfashion, and one or more analog-to-digital converters (ADCs) thatconvert the output signals generated by the amplifiers to digital form.For example, a matrix that includes a first amplifier having a firstinput and a first output, and a second amplifier having a second inputand a second output, a switch to couple the first output of the firstamplifier to the second input of the second amplifier, a first ADCcoupled to the first output of the first amplifier, and a second ADCcoupled to the second output of the second amplifier. In one embodiment,the signals detected by the antennas include magnetic resonance (MR)signals.

United States Patent Application Publication 2008/0084210 by Vaughan etal. published Apr. 10, 2008 titled “Multi-Current Elements for MagneticResonance Radio Frequency Coils” is incorporated herein by reference.Vaughan et al. disclose a current unit having two or more current pathsallows control of magnitude, phase, time, frequency and position of eachof element in a radio frequency coil. For each current element, thecurrent can be adjusted as to a phase angle, frequency and magnitude.Multiple current paths of a current unit can be used for targetingmultiple spatial domains or strategic combinations of the fieldsgenerated/detected by combination of elements for targeting a singledomain in magnitude, phase, time, space and frequency.

United States Patent Application Publication 2008/0129298 by Vaughan etal. published Jun. 5, 2008 titled “High field magnetic resonance” isincorporated herein by reference. Vaughan et al. disclose, among otherthings, multi-channel magnetic resonance using a TEM coil.

An article co-authored by one inventor of the present invention istitled “Imaging radio frequency electron-spin-resonance spectrometerwith high resolution and sensitivity for in vivo measurements” by HowardHalpern et al., Rev. Sci. Instrum. 60(6), June 1989, {40. Halpern, 1989#89} was attached as Appendix B to U.S. Provisional Patent Application61/306,917 titled “High-isolation transmit/receive surface coils andmethod for EPRI” filed Feb. 22, 2010 by Howard J. Halpern, which isincorporated herein by reference. Halpern et al. describe a radiofrequency (RF) electron-spin-resonance spectrometer with high molarsensitivity and resolution. 250-MHz RF is chosen to obtain goodpenetration in animal tissue and large aqueous samples.

Another article co-authored by inventors of the present invention istitled “A Versatile High Speed 250-MHz Pulse Imager for BiomedicalApplications” by Boris Epel, Sundramoorthy, S. V., Mailer, C. & Halpern,H. J. at the Center for EPR Imaging In Vivo Physiology, Department ofRadiation and Cellular Oncology, University of Chicago, Chicago, Ill.60637 (Concepts Magn. Reson. Part B (Magn. Reson. Engineering) 33B:163-176, 2008) {46. Epel, 2008 #2200} was attached as Appendix A to U.S.Provisional Patent Application 61/306,917 titled “High-isolationtransmit/receive surface coils and method for EPRI” filed Feb. 22, 2010by Howard J. Halpern, which is also incorporated herein by reference.Epel et al. describe a versatile 250-MHz pulse electron paramagneticresonance (EPR) instrument for imaging of small animals. Flexible designof the imager hardware and software makes it possible to use virtuallyany pulse EPR imaging modality. A fast pulse generation and dataacquisition system based on general purpose PCI boards performsmeasurements with minimal additional delays. Careful design of receiverprotection circuitry allowed those authors to achieve very highsensitivity of the instrument. In this article, they demonstrate theability of the instrument to obtain three-dimensional (3D) images usingthe electron-spin echo (ESE) and single-point imaging (SPI) methods. Ina phantom that contains a 1 mM solution of narrow line (16 μT,peak-to-peak) paramagnetic spin probe, their device achieved anacquisition time of 32 s per image with a fast 3D ESE imaging protocol.Using an 18-min 3D phase relaxation (T_(2e)) ESE imaging protocol in ahomogeneous sample, a spatial resolution of 1.4 mm and a standarddeviation of T_(2e) of 8.5% were achieved. When applied to in vivoimaging this precision of T_(2e) determination would be equivalent to 2Torr resolution of oxygen partial pressure in animal tissues.

U.S. Pat. No. 4,812,763 to Schmalbein issued Mar. 14, 1989 titled“Electron spin resonance spectrometer”, and is incorporated herein byreference. Schmalbein describes an electron spin resonance spectrometerthat includes a resonator containing a sample and arranged in a magneticfield of constant strength and high homogeneity. A microwave bridge canbe supplied with microwave energy in the form of an intermittent signal.Measuring signals emitted by the resonator are supplied to a detectorand a signal evaluation stage. A line provided between a microwavesource and the microwave bridge is subdivided into parallelpulse-shaping channels, one of them containing a phase shifter, anattenuator and a switch for the signal passing through the pulse-shapingchannels. In order to be able to set, if possible, an unlimitedplurality of pulse sequences for experiments of all kinds, thepulse-shaping channels are supplied in equal proportions from the lineby means of a divider. All pulse-shaping channels are provided with aphase shifter and an attenuator. The pulse-shaping channels arere-united by means of a combiner arranged before the input of a commonmicrowave power amplifier.

U.S. Pat. No. 6,639,406 to Boskamp, et al. issued Oct. 28, 2003 titled“Method and apparatus for decoupling quadrature phased array coils”, andis incorporated herein by reference. Boskamp, et al. describe a methodand apparatus for combining the respective readout signals for a loopand butterfly coil pair of a quadrature phased array used for magneticresonance imaging. The technique used to combine the signals introducesa 180-degree phase shift, or multiple thereof, to the loop coil signal,thereby allowing the loop coil signal to be decoupled from other loopcoil signals by a low-input-impedance preamplifier in series with thesignal. This patent describes a surface coil that is applied to onesurface of the body part being examined.

U.S. Pat. No. 7,659,719 to Vaughan, et al. issued Feb. 9, 2010 titled“Cavity resonator for magnetic resonance systems”, and is incorporatedherein by reference. Vaughan, et al. describe a magnetic resonanceapparatus that includes one or more of the following features: (a) acoil having at least two sections, (b) the at least two sections havinga resonant circuit, (c) the at least two sections being reactivelycoupled or decoupled, (d) the at least two sections being separable, (e)the coil having openings allowing a subject to see or hear and to beaccessed through the coil, (f) a cushioned head restraint, and (g) asubject input/output device providing visual data to the subject, theinput/output device being selected from the group consisting of mirrors,prisms, video monitors, LCD devices, and optical motion trackers. Thispatent describes a volume head coil that surrounds a human head.

U.S. Pat. No. 5,706,805 Swartz, et al. issued Jan. 13, 1998 titled“Apparatus and methodology for determining oxygen tension in biologicalsystems”, and is incorporated herein by reference. Swartz, et al.describe apparatus and methods for measuring oxygen tensions inbiological systems utilizing physiologically acceptable paramagneticmaterial, such as India ink or carbon black, and electron paramagneticresonance (EPR) oximetry. India ink is introduced to the biologicalsystem and exposed to a magnetic field and an electromagnetic field inthe 1-2 GHz range. The EPR spectrum is then measured at the biologicalsystem to determine oxygen concentration. The EPR spectrum is determinedby an EPR spectrometer that adjusts the resonator to a single resonatorfrequency to compensate for movements of the biological system, such asa human or animal. The biological system can also include other in vivotissues, cells, and cell cultures to directly measure pO₂non-destructively. The paramagnetic material can be used non-invasivelyor invasively depending on the goals of the pO₂ measurement. A detectinginductive element, as part of the EPR spectrometer resonator, is adaptedrelative to the measurement particularities.

U.S. Pat. No. 5,865,746 to Murugesan, et al. issued Feb. 2, 1999 titled“In vivo imaging and oxymetry by pulsed radiofrequency paramagneticresonance”, and is incorporated herein by reference. Murugesan et al.describe a system for performing pulsed RF FT EPR spectroscopy andimaging includes an ultra-fast excitation subsystem and an ultra-fastdata acquisition subsystem. Additionally, method for measuring andimaging in vivo oxygen and free radicals or for performing RF FT EPRspectroscopy utilizes short RF excitations pulses and ultra-fastsampling, digitizing, and summing steps.

U.S. Pat. No. 4,280,096 to Karthe, et al. issued Jul. 21, 1981 titled“Spectrometer for measuring spatial distributions of paramagneticcenters in solid bodies”, and is incorporated herein by reference.Karthe, et al. describe a spectrometer in which gradient coils areprovided in order to create an inhomogeneous magnetic field for use inanalyzing individual regions within the sample under examination. Thegradient coils and the modulating coils are operated by discrete pulses,rather than continuously. A keying unit coordinates the interaction ofthe various components of the spectrometer in order to monitor resonanceof the sample under examination while such pulses occur.

U.S. Pat. No. 5,828,216 to Tschudin, et al. issued Oct. 27, 1998 titled“Gated RF preamplifier for use in pulsed radiofrequency electronparamagnetic resonance and MRI”, and is incorporated herein byreference. Tschudin et al. describe a gated RF preamplifier used insystem for performing pulsed RF FT EPR spectroscopy and imaging or MRI.The RF preamplifier does not overload during a transmit cycle so thatrecovery is very fast to provide for ultra-fast data acquisition in anultra-fast excitation subsystem. The preamplifier includes multiplelow-gain amplification stages with high-speed RF gates inserted betweenstages that are switched off to prevent each stage from overloadingduring the transmit cycle.

U.S. Pat. No. 4,714,886 to one of the present inventors, Howard Halpern,issued Dec. 22, 1987 titled “Magnetic resonance analysis of substancesin samples that include dissipative material”, and is incorporatedherein by reference. U.S. Pat. No. 4,714,886 describes magneticresonance images of the distribution of a substance within a sample thatare obtained by splaying a pair of magnetic field generating coilsrelative to each other to generate a magnetic field gradient along anaxis of the sample. In other aspects, electron spin resonance data isderived from animal tissue, or images are derived from a sample thatincludes dissipative material, using a radio frequency signal ofsufficiently low frequency.

There is a need for an improved apparatus and method ofelectron-spin-resonance spectrometry and/or imaging to non-invasivelyprovide images and/or other signal measurements representative ofparticular internal structures and processes in the human body, and tobe able to distinguish malignant tissue from healthy tissue.

SUMMARY OF THE INVENTION

The present invention provides a method and apparatus for improvedsignal-to-noise (S/N) measurements useful for electron paramagneticresonance imaging (EPRI), in situ and in vivo, using high-isolationtransmit/receive surface coils and a series of pulse sets of variouslytemporally spaced-apart and phase-shifted pulses of RF energy. In someembodiments, a plurality of measurements are taken and recorded, whereineach measurement is based on establishing a static (DC) magnetic fieldon an animal tissue (e.g., a magnetic field having a substantially fixeddirection and a gradient field strength in a section of volume of tissueof a living human), then transmitting a set of RF pulses that includes afirst pulse, a first delay, a second pulse, a second delay and a thirdpulse, and then receiving, processing, and storing the resultant RFsignals from the tissue sample. By varying the strength of the DCmagnetic field (e.g., by generating the magnetic-field gradient thatincreases the field strength in some areas of the tissue being measuredand/or decreases the field strength in other areas of the tissue beingmeasured) and varying the direction of the gradient relative to thetissue being measured (e.g., by electro-magnetically changing thedirection of the gradient, or by physically moving the patient relativeto the gradient), varying the durations of the first and second delays,and varying the amounts of phase shifts of the second and third pulsesrelative to the phase of the first pulse, and storing that data alongwith data based on the RF signal received from the sample, an image(e.g., two-dimensional (2D) sections in various orientations, or athree-dimensional (3D) image of a volume) of the section or volume oftissue can be derived by computer calculations and displayed. Forexample, in some embodiments, each measurement uses a pulse set of threeRF pulses having a selected temporal spacing, selected amplitudes, andvarious phase-shift amounts, for example: a first RF pulse (e.g., insome embodiments, a pulse of about 35-ns duration, which equals about 9cycles of about 250 MHz cycles) followed after a first variable delay bya second pulse (e.g., of about 9 cycles of substantially the samefrequency but having half the amplitude and a shifted phase relative tothe cycles of the first pulse; in some embodiments, the cycles of thesecond pulse are obtained from the same signal source as those of thefirst pulse but are either not phase shifted (which is equivalent tophase shifted by 0 degrees), phase shifted by 90 degrees, phase shiftedby 180 degrees, or phase shifted by 270 degrees, then followed, after asecond variable delay, by a third pulse of about 9 cycles ofsubstantially the same frequency but having the same amplitude as thefirst pulse and a variously shifted phase relative to the cycles of thefirst pulse. In some embodiments, the first pulse has a magnitude thatflips the electron-spin directions (the direction of magnetization) by180 degrees and is thus called a “pi pulse”; the second pulse has amagnitude that flips the electron-spin directions (the direction ofmagnetization) by 90 degrees and is thus called a “pi-over-two pulse”(or “pi/2 pulse”); and the third pulse has a magnitude that again flipsthe electron-spin directions (the direction of magnetization) by 180degrees and is thus called a “pi pulse”. In some embodiments, thesetriplet sets of excitation RF pulses are configured to result in signalsthat represent the T₁ relaxation of electron spin (in contrast to the T₂relaxation of electron spin) in order to be sensitive to the oxygencontent in the tissue being imaged. In some such embodiments, the fourpossible phase-shift amounts of the second pulse and the four possiblephase-shift amounts of the third pulse provide sixteen combinations ofphase shift amounts for each set of the first delay (between the firstand second pulses) and second delay (between the second and thirdpulses). In some embodiments, this provides improved SNR inmicro-environmental images that are representative of particularinternal structures in the human body and spatially resolved images oftissue/cell protein signals responding to conditions (such as hypoxia)that show the temporal sequence of certain biological processes, and, insome embodiments, that distinguish malignant tissue from healthy tissue.

In some embodiments, the durations of the three pulses in a triplet setare kept constant and the frequency of the carrier in each pulse arekept constant in order to maintain the same spectral content (i.e., theFourier transform of the pulses yields the same spectrum of frequenciesand relative strengths for those frequencies) for every pulse, while thetotal strength of some of the pulses is varied to obtain differentamounts of rotation of the electron spins and/or magnetic moment of thereporter molecules.

In some embodiments, the present invention further includes medicalprocedures, animal models, and biological agents (such as viral “TrojanHorse” constructs or other vectors) that facilitate the obtaining ofimages that distinguish different types of tissues or healthy tissuesfrom malignant or infected tissues, that show various spatially andtemporally resolved signaling, regulation, promotion and responses of,for example, signaling peptides, protein products.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A1 shows a schematic representation of an inversion-recovery pulsesequence 101 useful for measuring T₁, according to some embodiments ofthe present invention.

FIG. 1B1 shows a schematic representation of a conventionalsaturation-recovery-with-echo-detection pulse sequence 102 useful formeasuring T₁.

FIG. 1C1 shows a schematic representation of a 2pESE pulse sequence 103useful for measuring T₂, according to some embodiments of the presentinvention.

FIG. 1A2 shows another schematic representation of inversion-recoverypulse sequence 101 useful for measuring T₁, according to someembodiments of the present invention.

FIG. 1B2 shows another schematic representation of conventionalsaturation-recovery-with-echo-detection pulse sequence 102 useful formeasuring T₁.

FIG. 1C2 shows another schematic representation of 2pESE pulse sequence103 useful for measuring T₂, according to some embodiments of thepresent invention.

FIG. 1D is a perspective schematic representation of a gradient-fieldset 104 of electromagnet coils useful for measuring T₁, according tosome embodiments of the present invention.

FIG. 2 is a graph 201 of spectroscopic measurements of the effectivespin-packet linewidth versus spin-probe concentration.

FIG. 3A and FIG. 3B present 1/T₂ and 1/T₁ images, respectively, obtainedfrom a mouse tumor, presented on the same drawing sheet for comparison.

FIG. 3C is an enlarged view of the screenshot of the 1/T₂ images of FIG.3A.

FIG. 3D is an enlarged view of the screenshot of the 1/T₁ images of FIG.3B.

FIG. 4A shows an SFR ESE pulse sequence, wherein the repetition rate isvaried.

FIG. 4B shows an SFR SPI pulse sequence, wherein the repetition rate isvaried.

FIG. 4C shows a saturation-recovery-with-echo-detection (SR) pulsesequence, wherein the delay time T is varied.

FIG. 4D1 shows an IRESE pulse sequence using a pi/2-pi echo-detectingpulse pair, wherein the delay time T is varied.

FIG. 4D2 shows an IRESE pulse sequence with a pi/2-pi/2 echo-detectingpulse pair, wherein the delay time T is varied.

FIG. 4E shows an IRSPI pulse sequence, wherein the delay time T isvaried.

FIG. 4F shows an SE pulse sequence, wherein the delay time T is varied.

FIG. 4G shows a two-pulse electron-spin echo (2pESE) sequence measuringT₁ by varying repetition time.

FIG. 5A shows a selected slice T_(1e) image and histogram obtained usingSFR ESE.

FIG. 5B shows a selected slice T_(1e) image and histogram obtained usingSFR SPI.

FIG. 5C shows a selected slice T_(1e) image and histogram obtained usingIRESE.

FIG. 5D shows a selected slice T_(1e) image and histogram obtained usingIRSPI.

FIG. 5E shows a selected slice T_(1e) image and histogram obtained usingSE.

FIG. 5F shows a selected slice T_(2e) image and histogram obtained using2pESE.

FIG. 6A is a T_(2e) image obtained using a 2pESE sequence.

FIG. 6B is a T_(1e) image obtained using an IRESE sequence.

FIG. 7 is an EPR oxygen image of two planes of a mouse leg bearing anFSa fibrosarcoma.

DESCRIPTION OF PREFERRED EMBODIMENTS

Although the following detailed description contains many specifics forthe purpose of illustration, a person of ordinary skill in the art willappreciate that many variations and alterations to the following detailsare within the scope of the invention. Accordingly, the followingpreferred embodiments of the invention are set forth without any loss ofgenerality to, and without imposing limitations upon, the claimedinvention. Further, in the following detailed description of thepreferred embodiments, reference is made to the accompanying drawingsthat form a part hereof, and in which are shown by way of illustrationspecific embodiments in which the invention may be practiced. It isunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the present invention.

The leading digit(s) of reference numbers appearing in the Figuresgenerally corresponds to the Figure number in which that component isfirst introduced, such that the same reference number is used throughoutto refer to an identical component which appears in multiple Figures.Signals and connections may be referred to by the same reference numberor label, and the actual meaning will be clear from its use in thecontext of the description.

Section #1: T₁ Imaging

Virtually all pulse-EPR imaging in animal specimens has used sequencessensitive to transverse relaxation times of the electron spin probe, T₂.{110. Matsumoto, 2006}, {46. Epel, 2008 #2200}. T₂ is a measure of phasecoherence that takes place as a result of quantum-mechanical spin flipscaused by multiple interactions with the environment. As has beendemonstrated in nuclear-magnetic-resonance imaging and spectroscopy, andwater-proton MRI, T₂ relaxation is sensitive not only to the loss ofenergy by the spin system to the environment (spin lattice) but multipleother relaxation processes that can affect the phase coherence of themagnetization spins as well as the loss of energy. Among theserelaxation processes are interactions, in animal tissue, with oxygen, adi-radical with two unpaired electrons in its outermost orbital. Thisboth dephases the spins of the reporter spin system but results in lossof energy from the reporter spin system.

One of the major confounding and blurring effects of using T₂ imagingfor oxygen measurement is the confounding spin dephasing by one moleculeof the reporter system by another reporter molecule. These cannot bedistinguished in a T₂ measurement of oxygen dephasing of the reporterspin system.

T₂ measurements are also known as transverse-spin-relaxation time (orsometimes spin-spin relaxation time) measurements. T₁ measurements arealso known as longitudinal-spin-relaxation-time measurements. Inprinciple, T₁ only measures the loss of energy by the reporter spinsystem to the environment, also known as the lattice, and thus T₁ isalso called the spin-lattice relaxation time (SLR time), while T₂ isalso called spin-spin relaxation time. This requires that themechanism(s) that can relax the longitudinal spins are much morerestricted than those that relax the transverse spin component. T₁-spinrelaxation is thereby a more specific process. An example, in principle,is that when energy is transferred from one reporter spin molecule toanother, the reporter spin system loses no energy. There is norelaxation. Thus, the T₁ measurement should be far less sensitive toself interaction than the T₂ measurement.

The present invention has obtained the first T₁-based tissue-oxygenimage using T₁-sensitive inversion recovery using spin-echo detection ofthe spin recovery. This gives longer relaxation times, which willprovide an even more direct sensitivity to pO₂.

Two modalities for determination of T₁ are commonly used in pulse EPR:saturation and inversion recovery. In the first case, presented in theFIG. 1B1, the EPR transition is saturated with a long (t>>T₁) RF pulseand recovery of EPR signal is detected as a function of separationbetween this pulse and detection sequence (two pulse echo in this case).For inversion recovery (FIG. 1A1), a π-pulse (pi pulse) is used toinvert the magnetization. We have implemented the inversion-recoverymethod since it has a twice larger effect and does not requireadditional hardware. In some embodiments, for projection generation,which acts as a “read out” of the T₁ information, the present inventionuses the electron-spin echo (ESE)-detection sequence.

In an EPR system, the magnetic moment will generally align with the H₀static magnetic field (conventionally and as used herein the directionof H₀ is designated as the z direction), but will precess at an angularfrequency ω that is proportional to H₀, and there exists a particularangular frequency ω_(R) that is resonant for a particular magnitude ofH₀. If an alternating magnetic field H₁ cos(ωt) (designated H₁ forsimplicity, conventionally and as used herein the direction of H₁ isdesignated as the x direction) is applied orthogonal to the H₀ staticmagnetic field, where ω is at the resonant angular frequency ω_(R), amagnetic moment that was parallel to the H₀ static magnetic field willprecess in the y-z plane; that is, the magnetic moment will precess butalways remain perpendicular to the H₁ (x direction) alternating field,and thus will periodically be pointed in a direction opposite H₀. If awave train (a radio-frequency (RF) pulse) were applied at a magnitude Aand for a pulse duration t_(w) the magnetic moment will precess throughan angle θ=AH₁t_(w). If an RF pulse of a selected magnitude A and pulseduration t_(w) is applied such that θ=π (i.e., θ=180 degrees), the pulsewill invert the magnetic moment and such a pulse is called a π pulse(also called a 180-degree pulse). Further, if an RF pulse of a differentselected magnitude A′ and pulse duration t_(w) is applied such thatθ=π/2 (i.e., θ=90 degrees), the pulse will turn the magnetic moment fromthe z direction to the y direction and such a pulse is called a π/2pulse (also called a 90-degree pulse). Note that either or both themagnitude A and pulse duration t_(w) may be varied to achieve a givendesired rotation of the magnetic moment. (See Charles P. Slichter“Principles of Magnetic Resonance, Third Enlarged and Updated Edition”,Springer Berlin-Heidleberg, 1996, pp 20-24.) Other pulses of selectedmagnitudes A and pulse durations t_(w) may be used to achieve otheramounts of rotation of the magnetic moment, such as a 2π/3 pulse (alsocalled a 120-degree pulse).

FIG. 1A1 shows a schematic representation of an inversion-recovery pulsesequence 101 that includes a first π pulse (also called a pi pulse,inversion pulse or 180-degree pulse) 110, a first time delay “T” 111after first π pulse (pi pulse) 110, then a second π/2 pulse (pi/2 pulseor 90-degree pulse) 112, a second time delay “τ” 113 after second π/2pulse (pi/2 pulse) 112; then a third π pulse (pi pulse or 180-degreepulse) 114, a third time delay “τ” 115 after third π pulse (pi pulse)114; then a readout-stage measurement 116 of the resulting spin echo.This pulse sequence is referred to herein as an inversion recoverysequence with electron-spin echo detection, or IRESE. The dashed line127 shows signal amplitude (arbitrary units; not to scale) of the spinecho as a function of time. In both cases (FIG. 1A1 and FIG. 1B1), thedetection sequence consists of two ESE pulses: a π/2 pulse (pi/2 pulseor 90-degree pulse) and a π pulse (pi pulse or 180-degree pulse). Notethat the durations of the pulses as shown in FIG. 1A1, FIG. 1B1, andFIG. 1C1 are not to scale with the times between pulses or times betweenpulse sequences. In some embodiments, the pulse durations are chosen as35 ns (nanoseconds) each (substantially square pulses of about 9 cyclesof a 250 MHz carrier wave), while the time τ 113 between the π/2 pulse112 and the π pulse 114 is chosen as τ=630 ns, and T 111 is varied for agiven set of signal acquisitions at eight values, denoted as variabledelay or VD in the IRESE section of Table 1 below, logarithmicallyspaced between 0.5 μs (500 ns) and 16 μs (16,000 ns). Further, themagnitudes of the readout signals 116, 126, and 136 are not to scalewith the magnitude of the excitation pulses 110, 112, 114, 120, 122,124, 132, or 134.

Pulse 112 and pulse 114 are also referred to as “readout pulses”. Insome other embodiments, rather than using a π/2 pulse (90-degree pulse)and a π pulse (180-degree pulse) as the readout pulses, two pulses, eachbeing a 2π/3 pulse (120-degree pulse), are used instead as the readoutpulses for an inversion-recovery measurement of T₁. The original spinecho measurements of Erwin Hahn, which were generated by two π/2 pulses,could also be used for the readout of the magnetization after an initialinversion pulse

FIG. 1B1 shows a schematic representation of asaturation-recovery-with-echo-detection (SR) pulse sequence 102 thatincludes a first saturation pulse 120, a first time delay “T” 111 afterfirst saturation pulse 120, then a second π/2 pulse (pi/2 pulse or90-degree pulse) 122, a second time delay “τ” 123 after the second pulse122; then a third π pulse (pi pulse or 180-degree pulse) 124, a thirdtime delay “τ” 125 after the third pulse 124; then a readout-stagemeasurement 126 of the resulting spin echo. The dashed line 127 showsrelative signal amplitude (arbitrary units; not to scale) of the spinecho as a function of time.

FIG. 1C1 shows a schematic representation of a 2pESE pulse sequence 103useful for measuring T₂, wherein pulse sequence 103 includes a first π/2pulse 132, a first time delay “τ” 133 after the first pulse 132; then asecond π pulse 134, a second time delay “τ” 135 after the second pulse134; then a readout-stage measurement 136 of the resulting spin echo.The delay time T_(R) 139 is the time between the end of onesignal-acquisition phase (e.g., of signal 136) and the start of the nextfirst pulse 132. The dashed line 127 shows signal amplitude (arbitraryunits; not to scale) of the spin echo as a function of time.

FIG. 1A2 shows another schematic representation of inversion-recoverypulse sequence 101 useful for measuring T₁, according to someembodiments of the present invention. Note that the durations of thepulses as shown in FIG. 1A2, FIG. 1B2, and FIG. 1C2 are more to scalewith the times between pulses or times between pulse sequences. In someembodiments, as described above, the pulse durations are chosen as 35 ns(nanoseconds) each (substantially square pulses of about 9 cycles of a250 MHz carrier wave), while the time τ 113 between the π/2 pulse 112and the π pulse 114 is chosen as τ=630 ns, and T 111 is varied for agiven set of signal acquisitions at eight values, denoted as VD in theIRESE section of Table 1 below, logarithmically spaced between 0.5 μs(500 ns) and 16 μs (16000 ns). Further, the magnitudes of the readoutsignals 116, 126, and 136 are not to scale with the magnitude of theexcitation pulses 110, 112, 114, 120, 122, 124, 132, or 134.

FIG. 1B2 shows another schematic representation of conventionalsaturation-recovery-with-echo-detection pulse sequence 102 useful formeasuring T₂, where the durations of the pulses and the times betweenpulses are more to scale than in FIG. 1B1.

FIG. 1C2 shows another schematic representation of 2pESE pulse sequence103 useful for measuring T₁, according to some embodiments of thepresent invention, where the durations of the pulses and the timesbetween pulses are more to scale than in FIG. 1C1.

FIG. 1D is a perspective schematic representation of a gradient-fieldset 104 of electromagnet coils useful for measuring T₁, according tosome embodiments of the present invention. In some embodiments, theplanes of all of the rectangular coils (coil 142LT (the left-hand topcoil in FIG. 1D), coil 142RT (the right-hand top coil in FIG. 1D), coil142LB (the left-hand bottom coil in FIG. 1D), coil 142RB (the right-handbottom coil in FIG. 1D), coil 143FT (the front top coil in FIG. 1D),coil 143BT (the back top coil in FIG. 1D), coil 143FB (the front bottomcoil in FIG. 1D), coil 143BB (the back bottom coil in FIG. 1D), areparallel to one another and these planes in turn are parallel to theplanes of the round coils. This is to make the main direction of themagnetic fields of each given strength in the region of interestparallel. In some embodiments, the planes of the pairs of similarlyoriented square coils, for example, above the horizontal plane, areoffset a little bit so that these rectangular coil pairs can be movedcloser to each other without bumping each other. In some embodiments,the distances between members of each pair are reduced to make thegradient (the amount of change in magnetic field strength) more uniform.

In some embodiments, the currents in each coplanar pair move in oppositesenses. For example, in the embodiment shown in FIG. 1D, the current incoil 141T is clockwise such that the differential field 151T is downward(subtracting from the main field 150 on the top side), while the currentin coil 141B is counterclockwise such that the differential field 151Bis up (adding to the main field 150 on the back side). By changing themagnitude of the currents in coils 141T and 141B the amount of gradientcan be varied. Similarly, in the embodiment shown, the current in coils142LT and 142LB are clockwise such that the differential field 152L isdownward (subtracting from the main field 150 on the left-hand side),while the current in coils 142RT and 142RB are counterclockwise suchthat the differential field 152R is upward (adding to the main field 150on the right-hand side); and the current in coils 143FT and 143FB arecounterclockwise such that the differential field 153F is upward (addingto the main field 150 on the front side), while the current in coils143BT and 143BB are clockwise such that the differential field 153B isdownward (subtracting from the main field 150 on the back side). Bychanging the directions and the relative and absolute amounts of currentin the three sets of coils, the gradient-field direction and magnitudecan be changed in plus and/or minus X, Y and/or Z directions.

In some embodiments of the gradient-field coils, there are left andright coplanar pairs (142LB and 142RB) that face the corresponding coils(142LT and 142RT) above the horizontal midplane, and front and backcoplanar pairs (143FB and 143BB) of coils below the horizontal midplanethat face the corresponding coils (143FT and 143BT) above the horizontalmidplane. In some embodiments (not shown here), for each pair of thegradient-field coils (142LB and 142RB), (142LT and 142RT), (143FB and143BB) (143FT and 143BT) there are an outside set of two pairs and aninside set of two pairs, one set closer—the other set farther apart. Thecoils that face each other across the horizontal have their currents inthe same direction. FIG. 2 presents a graph 201, where plot 211 shows1/T₁, the longitudinal relaxation rate of the magnetization described interms of magnetic-field linewidth units to which it is proportionalversus spin-probe concentration as measured using an inversion-recoverypulse sequence 101, and plot 212 shows 1/T₂, the transverse relaxationrate of the magnetization described in terms of a magnetic fieldlinewidth to which it is proportional versus spin-probe concentration asmeasured using a spin echo pulse sequence decay 103.

FIG. 2 presents a graph 201 of spectroscopic measurements of a theeffective spin-packet linewidth versus spin-probe concentration ofdeoxygenated samples of spin probe (OX063 radicalmethyl-tris[8-carboxy-2,2,6,6-tetrakis[2-hydroxyethyl]benzo[1,2-d:4,5-d′]bis[1,3]dithiol-4-yl]-trisodiumsalt, molecular weight=1,427 from GE Healthcare, Little Chalfont,Buckinghamshire, United Kingdom). In some embodiments, the solvent forthe spin probe is saline. All measurements are made at the same oxygenpartial pressure, 0 torr. Plot 211 shows the T₁ spin-packet linewidthversus spin-probe concentration as measured using an inversion-recovery(IRESE) pulse sequence 101 (see FIG. 1A1) according to the presentinvention, while plot 212 shows the T₂ spin-packet linewidth versusspin-probe concentration as measured using asaturation-recovery-with-echo-detection pulse sequence 102 (see FIG.1B1). Note that at low spin-probe concentrations, the T₁ and T₂measurements give approximately the same linewidth. However, asconcentration of spin probe rises, the additional confounding width ofthe T₁ measurements is reduced, and is only about one-fifth (⅕) that ofthe T₂ measurements. This is a remarkable reduction in the sensitivityof linewidth (or relaxation time) to the confounding variation of theconcentration of spin probe. That is, the linewidth increase (i.e.,error and/or uncertainty due to spin-probe concentration increase) isabout five times worse at high spin-probe concentrations if measuring T₂using saturation-recovery-with-echo-detection pulse (SR) sequence 102than if measuring T₁ using an inversion-recovery pulse (IRESE) sequence101.

FIG. 3A and FIG. 3B present T₂ and T₁ images obtained from the sametumor, presented on the same drawing sheet for comparison. FIG. 3C is anenlarged view of the screenshot of FIG. 3A, and FIG. 3D is an enlargedview of the screenshot of FIG. 3B.

FIG. 3A and FIG. 3C each show, at different magnifications, the sameorthogonal T₂ EPR image planes 301, 302 and 303 (labeled YX, ZY and YZrespectively) from an C57/B mouse leg bearing a syngeneic B16 melanoma.The red contour line in each view is that of the tumor from obtainedfrom a spatially registered MRI (a conventional nuclear magneticresonance image). Spin-packet linewidth derived pO₂ from the whole legare histogrammed in blue plot 304 and those only from the tumor are inred plot 305.

FIG. 3B and FIG. 3D each show, at different magnifications, the sameorthogonal T₁ EPR image planes 306, 307 and 308 (labeled YX, ZY and YZrespectively) from the same C57/B mouse leg bearing the syngeneic B16melanoma. The red contour line in each view is that of the tumor from aconventional nuclear MRI. Spin-packet linewidth derived pO₂ from thewhole leg are histogrammed in blue plot 309 and those only from thetumor are in red plot 310.

From the histograms 304, 305 of the pO₂ values derived from the T₂images and the histograms 304, 305 of the pO₂ values derived from the T₁images one can see a clear separation of the hypoxic tumor pO₂₅ in theT₁ image which is blurred in the T₂ image. This is due to the reducedconfounding effect of spin probe concentration on the images.

Section #2: Further T₁ Imaging Techniques—

a. Significance—Oxygen, HIF1α, VEGF and cancer biology: Regions of lowpO₂-hypoxia—are characteristic of solid tumors and have long been knownto increase resistance of malignant cells to radiation {1. Hall, 2000#1012; 2. Gatenby, 1988 #21; 3. Brizel, 1996 #695; 4. Brizel, 1999#1121; 5. Brizel, 1996 #1124; 6. Brizel, 1997 #1123; 7. Hockel, 1996#1111} and can be exploited for cancer therapy. 18. Shibata, 2002#1657.1 Hypoxia selects for a mutagenic, carcinogenetic, and aggressivemalignant phenotype. 19. Graeber, 1996 #942.1 Oxygen status is soimportant in tissue homeostasis that the absence of oxygen, hypoxia, isthe causative element in an entire regulatory peptide signaling cascade.Hypoxia inducible factor 1α, HIF1α, is the master regulator of theresponse of the cell response to hypoxia, initiating the signal cascade.{10. Semenza, 1998 #1693.} The cascade generates compensatory responsesto hypoxia at the cellular level, an intracrine response e.g.,apoptosis, {11. Carmeliet, 1998 #1132}, local vascular response throughincreased production of Vascular Endothelial Growth Factor (VEGF), aparacrine response {124. Carmeliet, 2000 #1131; 10. Semenza, 1998#1693}, and a general organism response, e.g., erythrocyte productionthrough the increase in production of erythropoietin, an endocrineresponse {10. Semenza, 1998 #1693.}

The anti-cancer success of anti-VEGF therapy {128. Sandler, 2007 #2192}as well as in vitro work, e.g., {126. Li, 2007 #2214; 125. Forsythe,1996 #2109} argues that in vivo, in situ, the signal peptide response tohypoxic environment is different in normal and malignant cells andtissues. The present invention, for the first time, uses electronparamagnetic resonance (EPR) imaging (EPRI) to demonstrate thisnon-invasively in animals with possible extension to humans. Most testsof cell signaling in vivo model subjected the whole animal to anenvironmental change and then imaged the local response ex vivo. {127.Picchio, 2008 #2103.} There is virtually no literature dealing with theheterogeneity in normal tissue and solid tumors. Although in vitro workhas established HIF1α signaling, this has profound implications fortherapeutic strategy. For example, it may explain failure of oxygenmanipulation to enhance tumor therapy with radiation. {129. Suit, 1984#1584}

A major goal of the Center for EPR Imaging In Vivo Physiology at theUniversity of Chicago is obtaining uniquely high-resolution quantitativeimages of tissue and tumor pO₂. The present invention provides anentirely new means of molecular imaging of the peptide signal responseto hypoxia, using EPR, registered with pO₂ images. This technique can beextended to a vast array of peptide-signaling processes. An example ofthis is the imaging of VEGF, a HIF1α cascade signal response to createnew vessels. Combined, co-localized images of pO₂ and peptide-signalingresponse will produce a quantified, localized relationship between theextent of hypoxia and the cell/tissue signal response to hypoxia invivo, in situ, which we hypothesize is different in malignant and normaltissue. We anticipate the eventual extension of EPR imaging technologyto humans. pO₂ stimulus images registered with peptide signal responsewill show individual variations in local stimulus-response to guideindividual therapy. The cell signaling technology described here willimpact the study of human health and disease.

FIG. 8 is an EPR oxygen image of two planes of a mouse leg bearing anFSa fibrosarcoma. Colorbars show pO₂ in mm/hg (torr). Numbers on planesare mm. Resolution: 1 mm spatial, 3 torr pO₂. Tumor is not distinguishedin this image but separately defined using a registered T₂ MRIindicating large central oxygen gradients in the tumor.

Molecular imaging shows heterogeneity of tumor/tissue condition andsignal response: Although cell signaling discoveries have providedunique insight into modes by which cells communicate with cells in theirenvironment, {14. Alberts, 2008 #2096} these studies of isolated cellsin artificial homogeneous environments contrast with the enormousheterogeneity of a living animal as shown in FIG. 4 and {15. Fischbach,2009 #2089}. The interaction of anatomy and signaling molecules throughvascular bed structure, target organ distance, size and location canaffect signaling. Organ- or tissue-dependent modulation of the signalingcan provide another layer of control that needs to be understood tofully comprehend the physiology of signaling. A crucial reason to imagecell signaling is this variation with position of cellular environments,shown in FIG. 1 . Registering images of a quantified environmentcharacteristic like pO₂ with images of the peptide response allows thedevelopment of models stimulus and response in a native environment.

Reporter protein (RP)/molecular beacon (MB) imaging technologies: Thereporter gene LacZ has been a major tool used to dissect transcriptioninduction using optical and fluorescence molecular beacon (MB)detection. {16. Alam, 1990 #1662.} The original such technology, LacZ,the bacterial gene encoding β-galactosidase (reporter) turns the indolelinked sugar X-Gal blue, an optical MB. {Holt, 1958 #1663.} By couplingthe β-galactosidase gene to a gene of interest, gene expression isdirectly seen taking place in blue cells. Many other such technologieshave followed. {16. Alam, 1990 #1662; 18. Chalfie, 1994 #1664; 19.Weissleder, 2003 #1694; 20. McCaffrey, 2003 #1692; 21. Blasberg, 2003#1681; 22. Massoud, 2003 #1687; 23. Herschman, 2003 #1684}, producingchromophore or fluorescent MBs in cells producing transcriptionallycoupled gene products that can be detected and imaged in vivo. The workproposed in this grant uses this basic technique, modified to turn on anEPR MB in response to hypoxia simultaneously imaged in vivo, obviatingthe problems with radionuclide, optical or MRI techniques.

Molecular Imaging Techniques Other than EPR Do Not Easily Allow Imagingof Animal Environment Condition and Cell Signal Response: Optical imagesand radionuclide imaging dominate molecular imaging. {24. Dothager, 2009#2227.} Optical techniques (e.g., www.xenogen.com/prodimagl.html) usereporter genes that can be engineered into transgenic mice {25. Zhang,2001 #1695} or into implanted tumor cells in mice {26. Adams, 2002#1680} are surface weighted because of the rapid non-resonant absorptionof optical frequency light by tissue. This makes it difficult toquantify image signal intensity, linewidths or relaxation times of depthgreater than a few mm. {27. Kirkpatrick, 2004 #1691.} Other than inartificial systems such as window chamber {28. Dewhirst, 1996 #1775},quantified relationship between stimulus such as micro-environmentaloxygen and peptide signal response is difficult.

Detection of radiotracer with positron emission tomography (PET) avoidsproblems with depth sensitivity {29. Schober, 2009 #2099; 30. Sun, 2001#1374; 31. Blasberg, 2003 #1665} and is extremely flexible. Theadvantage of radiotracer reporters is that it can be immediatelytranslated to human studies. However, the major problems with PETimaging is its limited resolution in space (˜2 mm) and in time anddistinguishing signal from the environmental stimulus reporter from thepeptide signal response reporter. For radionuclide studies, hypoxia isdefined as the reductive retention of nitro-imidazole {33. Raleigh, 1992#765; 34. Evans, 1996 #931} or ATSM copper chelates. {35. Lewis, 1999#1371.} Hypoxic signaling via HIF1α might be imaged as is proposed inthis grant with vectors containing hypoxia responsive elements (HREs)that bind HIF1α promoting production of, e.g., a thymidine kinase RPthat would cause hypoxic cells to retain radioactive thymidine (themolecular beacon (MB)). This has severe limitations:

Firstly, it is difficult to distinguish the signal from the compoundsignaling hypoxia from the thymidine retained through phosphorylation,signaling hypoxic response. EPR allows spectrally distinct hypoxiaimages and the peptide signal response images.

Secondly, the EPROI is quantitative while the reductive retention imageis qualitative. Radionuclide images depend heavily on access of theradionuclide to the location where oxygen is measured, and other aspectsof local tissue reductive capability, i.e., P450 reductase, xanthineoxidase, etc. activity. {36. Melo, 2000 #2229.} For EPROI, as long assome spin probe reaches the location, the oxygen measurement dependsonly weakly on the signal amplitude. Rather it depends on the signalrelaxation time or line width.

Molecular imaging with MRI creates contrast with the RP requiring anextremely large molecular signal {37. Louie, 2000 #1395; 38. Weissleder,2000 #1673} because the technology introduces contrast in a very highsignal background. Unlike MRI, the EPR technology activates a “beacon inthe dark”. In addition, MRI images provide poor pO₂ sensitivity.

EPR oxygen images use very low magnetic fields and are specific andsensitive to pO₂: EPR images are obtained at excitation RF frequenciesof very-high-field (6-7 T) whole-body MRIs {39. Vaughan, 2009 #2230} butbecause the magnetic moment of the electron is 658 times that of thewater proton, magnetic fields are 1/658 that of MRI. EPRI uses a lowfield with inexpensive magnet systems of about 90 Gauss=9 milliTesla(mT) at 250 MHz frequency. {40. Halpern, 1989 #89; 41. Halpern, 1991#899.} This is a low-cost technology not requiring superconductingmagnets, although standard-field MRI is, in some embodiments, used toidentify tumor. EPR spectral linewidths of certain carbon-centered spinprobes, trityls, are specific and sensitive to local pO₂. {42. Halpern,2003 #1798.} The narrow (μT) spectral linewidths, or, equivalently, theinverse transverse relaxation times (1/(5 μs)) of these spin probes aredirectly proportional to the local oxygen concentration. They give adirect quantitative readout of tissue micro-environmental pO₂. Usingspectroscopic EPR imaging {43. Lauterbur, 1984 #177; 44. Maltempo, 1986#181; 45. Halpern, 1994 #93; 46. Epel, 2008 #2200}, spatial images ofquantitative tissue pO₂ may be obtained from tumor and normal tissues ofliving animals.

Registered images of cell signaling will show local tissue response topO₂ stimulus. In some embodiments, the present invention obtainssimultaneous registered images of cell signals responding to low pO₂using nitrogen-centered molecular beacons activated byhypoxia-signaling-coupled reporter proteins. These cell-signal imageswould be spectrally distinct from the trityl-based pO₂ images and are,in some embodiments, obtained simultaneously with them. At 9 mT, carboncentered and the central manifold of ¹⁴N have sufficiently differentabsorption frequencies (˜8.4 MHz) that they are, effectively, two-colorimages. The readout frequencies are low enough to avoid poisonousnon-resonant absorption to allow oxygen quantification deep in livingtissue. {45. Halpern, 1994 #93.} A different embodiment withsimultaneous imaging of pO₂, HIF1α signaling and the vascularendothelial growth factor (VEGF) response to HIF1α would usecarbon-centered oxygen-sensitive trityl radicals and ¹⁴N and ¹⁵Nmolecular beacons, effectively providing three-color imagesautomatically registered with each other.

The present invention, for the first time, provides automaticco-localization of micro-environment stimulus and cell signal responsein native animal tissue and tumor environment, allowing theircomparison. Distinct responses of normal and tumor tissue will provideinsight into therapies that can exploit these differences, targetingmalignant tumors and sparing normal tissues. We believe this to beparadigm-shifting work, sharpening the in vivo understanding ofsignaling process. The present invention will allow monitoringsubject-to-subject and tumor-to-tumor variation, allowing a moreindividualized therapy. This will open a major avenue to the improvementof the therapeutic ratio for cancer therapy.

Section #3: T_(1e) Imaging Using Filtered Backprojection and SinglePoint Imaging Methodologies

Spin-lattice relaxation (T_(1e)) of a spin probe can be sensitive tovarious environmental parameters including local oxygen pO₂. We havedeveloped three dimensional pulse imaging of T_(1e) in vivo using fastrepetition time saturation, inversion recovery and, stimulated echosequences. T_(1e) images generated by sequences that have electron spinecho readout are reconstructed with filtered backprojection protocolswhile those using free induction decay readout are reconstructed withthe single point imaging protocols. We compared T_(1e) and T_(2e)imaging of narrow line trityl spin probe in vitro to find theirperformances very similar. However, for in vivo oxymetry T_(1e) imagingis found to be more promising due to weaker dependence of T_(1e) onenvironmental factors other than oxygen.

Introduction: Transverse relaxation or T_(2e) based EPR continuous waveand pulsed imaging techniques have proved to be very promisingmethodologies for oxygen imaging using injected paramagnetic molecule asthe probe of the spatial distribution of oxygen in animals {101. Halpern1989 #89}; {102. Kuppusamy 1994}; {103. Elas 2003}; {104. Mailer 2006};{105. Epel 2010}; {106. Subramanian 2002}. In many cases the samerelaxation mechanisms that affect T_(2e) of a spin probe, act onspin-lattice relaxation, T_(1e), making T_(1e) sensitive to theenvironment {107. Slichter, 1996}. For example, spin exchangeinteraction with molecular oxygen results in linear R_(2e)=1/T_(2e) andR_(1e)=1/T_(1e) dependence on pO₂ with nearly identical proportionalitycoefficients {108. Ardenkjaer-Larsen, 1998}. On the other hand, not allrelaxation mechanisms affect T_(2e) and T_(1e) in the same way. Forexample, the inter-molecule electron spin dipole interactions will notaffect spin-lattice relaxation while enhancing the phase relaxation.This makes T_(1e) imaging a very attractive imaging modality, separatefrom T_(2e) imaging. However, no systematic attempts to image in vivoT_(1e) using pulsed methods in vivo have been undertaken.

Given the widely used T_(2e) pulsed EPR oxygen imaging techniques, it isnatural to use similar techniques to read out T_(1e) based imageinformation. Although very different in imaging principles and observedrelaxation kinetics both electron spin echo (ESE) imaging oxymetry andsingle point imaging (SPI) oxymetry can precisely measure T_(2e). ESEimaging uses filtered backprojection (FBP) for image reconstruction froma number of static gradient projections recorded with fixed gradientamplitude, |{right arrow over (G)}|, and different gradientorientations. The projections are obtained by the Fourier transformationof time domain signals. In order to preserve correct phase informationdead-time free time domain signals are required which can be achieved bygeneration of echoes {104. Mailer, 2006}. In our earlier work we usedtwo pulse π/2-τ-π-τ-echo ESE (2pESE for brevity) {104. Mailer, 2006};{109. Epel, 2008 #2200}. To measure T_(2e) multiple separate images withdifferent τ delay values were acquired and the exponential decay timesof signal in each individual voxel in those images measured.

SPI methodology is based on different principles {106. Subramanian,2002}; {110. Matsumoto, 2006}. The dead time free acquisition isachieved by recording the single point on free induction decay, FID, ata known time, t_(SPI), as a function of stepped static gradientamplitudes sampled on a cubic grid. This signal forms a 3D“pseudo-echo”, the FT of which generates a spatial image. The spatialinformation is encoded into the phase of FID. In the simplest form ofSPI, the phase relaxation times are extracted from multiple imagesobtained at different t_(SPI). The fit of individual voxels toexponential decay gives the FID dephasing time T_(2e)* directly relatedto T_(2e) as 1/T_(2e)*=1/T_(2e) ^(hf)+1/T_(2e), where T_(2e) ^(hf) isthe oxygen independent phase relaxation due to hyperfine interactionwith trityl nuclei. More advance imaging schemes were developed toimprove the precision and reduce artifacts of SPI technique {110.Matsumoto, 2006}; {111. Devasahayam, 2007}.

For selection of a pulse method for T_(1e) imaging number of pulsesequence parameters should be taken into account. One of them is thepulse sequence bandwidth. Since the whole projection is acquired atonce, the bandwidth of the pulse sequence has to be broad enough tocover the equivalent bandwidth of the EPR line broadened by any appliedgradient. A bandwidth of about 5-10 MHz is typically sufficient for lowfrequency in vivo imaging of 3 cm specimens such as portions of mouseanatomy {109. Epel, 2008 #2200}. Another requirement is a dead time freeacquisition, which can be achieved by utilizing the same principlesimplemented in the ESE FBP T_(2e) imaging protocols.

-   -   We have selected three conventional ways to determine T_(1e),        two of which can be combined with two readout imaging        methodologies:        -   Saturation by fast repetition (SFR ESE and SFR SPI);        -   Inversion recovery (IRESE and IRSPI);        -   Stimulated echo (SE, ESE only).

In the SFR experiment (FIGS. 1A and 1B) the amplitude of the ESE or FIDis measured as a function of repetition time, T_(R), of the respectivesequence. A short repetition time (less than T_(1e)) saturates the spinsso the echo amplitude reduces as exp(−T_(R)/T_(1e)). Varying therepetition rate monitors the spin system saturation to get T_(1e). Thesecond type of T_(1e) sequence, inversion recover, inverts the spinpolarization using a broadband π pulse and the recovery is measuredusing as a function of the delay T after the inversion pulse. The echodetected inversion recovery sequence with ESE detection, IRESE (FIG.1A1) has been used for T_(1e) sensitive imaging {112. Eaton, 1987}. Thedelay τ was kept fixed during experiment. The bandwidth of this sequenceis approximately equal to the bandwidth of the 2 pulse ESE detectionsequence. The inversion recovery sequence for SPI, referred to as IRSPI,utilizes FID detection (FIG. 1D) and the bandwidth of this sequence isapproximately equal to the bandwidth of the inversion pulse. Analternative approach involves saturation recovery experiment. Here, theinversion pulse is replaced by long, t_(p)>>T_(1e), saturation pulse.The bandwidth of this pulse is insufficient for the purposes of imaging.Because additional facilities such as modulation of the magnetic fieldor pulse frequency during this pulse may be required to increase itsbandwidth, we excluded this sequence from our evaluation. The thirdsequence, SE (FIG. 1E), is the three-pulse ‘stimulated’ echo sequence{113. Schweiger, 2001}. In this sequence the π pulse of the 2 pulse ESEexperiment is split into two π/2 pulses separated by a waiting time T.After the first two π/2 pulses the magnetization is stored along thez-axis (longitudinal axis) where it remains during the time T. Thiswaiting time is varied allowing the magnetization to decay with T_(1e).The third π/2 pulse rotates the z-component back into the transversexy-plane where it gives rise to a stimulated echo at fixed time τ afterthe third pulse. This sequence is known to have nearly double bandwidthas compared to 2 pulse ESE (and thus IRESE) sequence with the samelength of the RF pulses {114. Kevan, 1990} and, therefore, is verypromising for reducing applied power in living subject.

We present a study of these different methods for imaging of T_(1e)using our 250 MHz pulse spectrometer. We also compare the precision ofT_(1e) imaging with that of T_(2e) imaging for determination of the O₂concentration in the phantoms.

Materials and Methods:

Spin Probe: The spin probe used for the EPR imaging was a OX063 radicalmethyl-tris[8-carboxy-2,2,6,6-tetrakis[2-hydroxyethyl]benzo[1,2-d:4,5-d′]bis[1,3]dithiol-4-yl]-trisodiumsalt, molecular weight=1,427 from GE Healthcare (Little Chalfont,Buckinghamshire, UK). The 1 mM solution of spin probe in saline wascontained in a flat-bottomed borosilicate glass cylinder of 9.5 mm innerdiameter and 45 mm length. The 0% O₂ sample was deoxygenated using amultiple-cycles freeze-pump-thaw technique and flame sealed. The 9.3% O₂sample was produced by bubbling of the solution with correspondingnitrogen-oxygen gas mixture and then was sealed with epoxy. Samples wereplaced into the resonator horizontally along the resonator's axis ofsymmetry and centered in the axial plane of the resonator. Becausesamples were half-full this produced a meniscus at the liquid-aircontact surface.

Pulse Imager and Pulse Sequences: In this work we used the versatilepulse 250 MHz imager described in details elsewhere {109. Epel, 2008#2200}. To utilize the full power of our 2 kW RF amplifier {115. Quine,2006} (Tomco Technologies, Norwood SA, Australia) the transmit-receiveswitch of the imager was redesigned using high power components andutilizing a new protection scheme {116. Sundramoorthy, 2009.} A pulseamplitude modulation switch was added to produce π/2- and π-pulses ofequal duration (hence equal bandwidth) {117. Quine, 2010}. The imagercontrol software SpecMan4EPR version 1.1.6 {118. Epel, 2005} was used.

To facilitate the image comparison, the measurement time was kept 10minutes for all images. Since pulse sequences are intended to imagesamples with heterogeneous relaxation times, we used the same sequencesfor phantoms and animal imaging. Standard deviation of relaxation timesin a homogeneous phantom was used as an estimation of relaxation timeerrors. Two outer layers of images were excluded from standard deviationcalculations to avoid partial volume averaging artifacts. Tables 1 and 2present parameters of T_(1e) and T_(2e) sequences. The repetition timefor ESE sequences, T_(R), was adjusted to keep the delay between thelast pulse in a sequence to the first pulse, T^(LF) _(R), of the nextsequence constant. This method of repetition time definition is found tobe more efficient as compared to conventional method where repetitiontime in the experiment is kept constant, independently of sequencelength. The optimal repetition time was determined by decreasing T_(R)from 5T_(1e) until sequence still provided correct values for T_(1e) orT_(2e). To accelerate acquisition of the IRESE/IRSPI images, the imagecorresponding to the last delay T was substituted with an image obtainedwithout inversion pulse and delay T. This halved the acquisition time ofthe last delay image.

For all ESE sequences the same 3D FBP protocol {104. Mailer, 2006};{119. Eaton 1991} was applied: 208 projections corresponding to the18×18 (eighteen-by-eighteen) equal solid angle gradient spacing {120.Ahn 2007(1)} were acquired; gradient strength was |{right arrow over(G)}|=15 mT/m; object field of view was 4.24 cm.

A baseline (acquisition at a far off-resonant field) acquired everyfourth trace (53 traces in all). To reduce FBP reconstruction artifactsthe acquired set of projections was four-fold linearly interpolated{121. Ahn 2007(2)} and filtered with a 3D Ram-Lak filter with a cutoffat 0.5 times the Nyquist frequency. In the images we kept only thosevoxels with a signal amplitude greater than 15% of the maximum amplitudeat the shortest delay. Further data acquisition and processing methodsare discussed in detail elsewhere {109. Epel, 2008 #2200}.

The SPI protocol involved acquisition of 5547 FIDs at delay t_(SPI)=1000ns with gradients corresponding to 23³ matrix in which only thegradients inside the sphere with the diameter of 23 gradients weretaken. The maximum gradient of 15 mT/m was used. A baseline was acquiredevery 20th trace to suppress imager related artifacts. The 3D‘pseudo-echo’ matrix was apodized with hamming window and Fouriertransformed to produce final image. All data processing was performedusing in-house MATLAB (The Mathworks, Inc., and Natick, Mass., USA)software.

Non-imaging versus imaging conditions: Acquisition of spatialinformation requires a considerable time. Therefore, imaging protocolshave to balance between precision of spatial and relaxation-timemeasurements. For example, the relaxation times are estimated from fiveto eight points on the decay curve. Moreover, the time of in vivoimaging can be limited by the animal physiology. These restrictions donot apply for non imaging measurements on phantoms, which can have largenumber of delays and can take much longer time. The parameters ofsequences for these measurements are selected so that they have noinfluence on measured relaxation times.

Animal Imaging: T_(1e) and T_(2e) images were sequentially taken on thesame animal with no delay in between. All animal experiments were doneaccording to the USPHS “Policy on Humane Care and Use of LaboratoryAnimals”, and the protocols were approved by the University of ChicagoInstitutional Animal Care and Use Committee. The University of ChicagoAnimal Resources Center is an Association for Assessment andAccreditation of Laboratory Animal Care-approved animal care facility.

Results: To demonstrate applicability of T_(1e) imaging for oxymetry wepresent the imaging results on two phantoms with different concentrationof O₂: 0% (Table 3, FIG. 2 ) and 9.3% (Table 4), respectively. Thisrange of oxygen concentrations is important hypoxia studies on animals{103. Elas 2003}; {122. Elas 2008}; {123. Matsumoto 2008}. Therelaxation times determined under non-imaging conditions (no appliedgradients, T_(R)=15T_(1e)) are given in the footnotes of the tables. Theresults of T_(2e) measurements using 2pESE are given in the tables forcomparison. The standard deviation of relaxation times in homogeneousphantom was used for estimation of errors. Between all T_(1e) imagingmethods IRESE sequence demonstrated the smallest standard deviation. SEshowed slightly worse performance, while SFR had very large standarddeviations on 0% O₂ phantom and was unable to produce T_(1e) image of9.3% O₂ phantom. 2pESE performance superseded the performance of allT_(1e) methods, however on 0% O₂ phantom the performance of IRESE camevery close to the performance of 2pESE. Similarly to ESE, IRSPI sequencedemonstrated much better performance than SFR SPI. Here we should notethat the comparison of absolute precisions of ESE and SPI methodologiesis outside of the scope of this work due to the lack of expertise inSPI.

For demonstration of the T_(1e) imaging on a live animal we selected theIRESE (FIG. 3A) methodology, which showed the best performance onphantoms, and compared the T_(1e) image with T_(2e) image (FIG. 3B)obtained using 2pESE on the same animal. The outlines and generalpatterns of the images are very similar. The average relaxation times inT_(2e) image are shorter, consistent with the typical ratios of T_(1e)and T_(2e) in trityls. Nevertheless the breadth of relaxation timehistograms is approximately the same.

Discussion: The data presented in the Results subsection demonstrate thefeasibility of T_(1e) imaging of live animals. IRESE images showcomparable with T_(2e) images quality both in vitro and in vivo. Themajor factor that affects precision of T_(1e)/T_(2e) imaging is an imagesignal to noise ratio (SNR). Assuming that for all methodologies theimager noise characteristics are equal, the image SNR will be governedby an amplitude of a signal and number of acquisitions. Inversionrecovery has the highest change in a signal amplitude due to relaxation,double of that for other sequences, since the evolution of signal fromnegative to positive is monitored. On the other hand the duration ofinversion recovery sequences is longer than T_(2e) sequences, whichreduces the number of sequence repetitions per unit time and hence SNR.The amplitude of signal for SFR is proportional to exp(−T_(R)^(MIN)/T_(1e)), where T_(R) ^(MIN) is the minimum T_(R) in anexperiment. In our instrument minimum T_(R) is governed by the dutycycle of the power amplifier, insufficient to generate SFR sequenceswith short enough T_(R). This makes SFR imaging not feasible for ourinstrument. The performance of SE method is worse than IRESE due totwice lower echo signal {114. Kevan, 1990}. However certain advantagescan be derived from considerably smaller power requirements of thissequence. SE image was taken using only 8% of RF power required forother methods excluding SFR SPI (see Table 2). Considering all thesefactors and nearly equal T_(1e) and T_(2e), inversion recovery methods,IRESE and IRSPI, should have comparable performance to 2pESE andstandard SPI images, which was demonstrated in the experiment.

The reduction in RF power requirements for SE sequence may make itattractive for large subject imaging. The power required for RF pulseswith identical B₁ is growing proportional to the resonator volume. Largeresonators may require more power than available sources can deliver.The lower average power deposition may also favor this sequence forhuman applications.

T_(1e) experiments can not deliver correct concentration map of thesample. Being acquired at non-zero τ, the T_(1e) amplitude image willalways carry an effect of T_(2e). To obtain true amplitude, the T_(2e)measurement has to be performed and amplitude extrapolated to time 0.Thus it might be of interest to combine inversion recovery T_(1e) andT_(2e) imaging into one experiment. Another advantage of such a jointexperiment would be an economy on delays since there will be no need tomeasure IRESE or IRSPI with long delay T—the result of such measurementis equal to experiment with no inversion pulse (2pESE or FID SPI).

Conclusions: T_(1e) imaging is feasible and comparable in precision withT_(2e) imaging. Therefore, it can be considered as an alternative methodfor oxymetry and other applications. Different applications andinstruments may benefit from different T_(1e) methods, with inversionrecovery imaging exhibiting the best relaxation time precision while SEimaging having the least power requirements.

FIGS. 4A-4F show various pulse sequences for determination of T_(1e):FIG. 4A shows an SFR ESE (saturation by fast repetition ESE) pulsesequence, wherein the repetition rate is varied, FIG. 4B shows an SFRSPI (saturation by fast repetition SPI) pulse sequence, wherein therepetition rate is varied, FIG. 4C shows an SR (saturation recovery)pulse sequence, wherein the delay time T is varied, FIG. 4D1 shows anIRESE (inversion recovery with ESE detection) pulse sequence, whereinthe delay time T is varied, FIG. 4E shows an IRSPI (inversion recoverywith SPI detection) pulse sequence, wherein the delay time T is varied,and FIG. 4F shows an SE (stimulated echo) pulse sequence, wherein thedelay time T is varied.

Regarding FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F: theselected slices and histograms of T1e images are obtained using for FIG.5A—SFR ESE; for FIG. 5B—SFR SPI; for FIG. 5C—IRESE; for FIG. 5D—IRSPI;for FIG. 5E—SE; and for FIG. 5F—2pESE. FIG. 5F shows the slice andhistogram of T_(2e) 2 pESE image. Experiments A for FIG. 5A, C for FIG.5C, E for FIGS. 5E, and F for FIG. 5F are performed on 1 mM sample 0%O₂, experiments B for FIGS. 5B and D for FIG. 5D are performed on 3 mM0% O₂ sample of triarylmethyl radical OX063.

FIG. 6A and FIG. 6B show T_(2e) and T_(1e) images obtained using 2pESEand IRESE pulse sequences, respectively.

Tables

TABLE 1 Pulse sequences. Description π/2-τ-π-τ-echo; 35 ns π/2 and π RFpulses; τ = 630 ns; 16-step phase cycling, 16640 echoes, including phasecycling; 8 images with different repetition times logarithmically spacedbetween 10 μs and 25 μs; imaging time 10 minutes. First First SecondSecond Detection Detection Protocol N pulse delay pulse delay channel Rechannel Im SFR 1 0.5X 630 ns  X 630 ns  A  B ESE 2 0.5X 630 ns −X 630 ns A  B 3 0.5X 630 ns  Y 630 ns −A −B 4 0.5X 630 ns −Y 630 ns −A −B 5−0.5X 630 ns  X 630 ns −A −B 6 −0.5X 630 ns −X 630 ns −A −B 7 −0.5X 630ns  Y 630 ns  A  B 8 −0.5X 630 ns −Y 630 ns  A  B 9 0.5Y 630 ns  X 630ns  B −A 10 0.5Y 630 ns −X 630 ns  B −A 11 0.5Y 630 ns  Y 630 ns −B  A12 0.5Y 630 ns −Y 630 ns −B  A 13 −0.5Y 630 ns  X 630 ns −B  A 14 −0.5Y630 ns −X 630 ns −B  A 15 −0.5Y 630 ns  Y 630 ns  B −A 16 −0.5Y 630 ns−Y 630 ns  B −A π-T-π/2-τ-π-τ-echo; 35 ns π/2 and π RF pulses; τ = 630ns; 16-step phase cycling applied only for detection sequence, 7520acquisitions per T, including phase cycling; 8 T's denoted as VD in thetable below logarithmically spaced between 0.5 μs and 16 μs; T^(LF) _(R)= 25 μs; imaging time 10 minutes. First First Second Second Third ThirdDetection Detection N pulse delay pulse delay pulse delay channel Rechannel Im IRESE 1 X VD 0.5X 630 ns  X 630 ns  A  B 2 X VD 0.5X 630 ns−X 630 ns  A  B 3 X VD 0.5X 630 ns  Y 630 ns −A −B 4 X VD 0.5X 630 ns −Y630 ns −A −B 5 X VD −0.5X  630 ns  X 630 ns −A −B 6 X VD −0.5X  630 ns−X 630 ns −A −B 7 X VD −0.5X  630 ns  Y 630 ns  A  B 8 X VD −0.5X  630ns −Y 630 ns  A  B 9 X VD 0.5Y 630 ns  X 630 ns  B −A 10 X VD 0.5Y 630ns −X 630 ns  B −A 11 X VD 0.5Y 630 ns  Y 630 ns −B  A 12 X VD 0.5Y 630ns −Y 630 ns −B  A 13 X VD −0.5Y  630 ns  X 630 ns −B  A 14 X VD −0.5Y 630 ns −X 630 ns −B  A 15 X VD −0.5Y  630 ns  Y 630 ns  B −A 16 X VD−0.5Y  630 ns −Y 630 ns  B −A π/2-τ-π/2-T-π/2-τ-echo; 60 ns RF pulses; τ= 550 ns; 32-step phase cycling, 12160 acquisitions per T, includingphase cycling; 8 T's denoted as VD in the table below logarithmicallyspaced between 0.45 μs and 7 μs; repetition time 20 μs; imaging time 10minutes. First First Second Second Third Third Detection Detection Npulse delay pulse delay pulse delay channel Re channel Im SE 1 0.5X 630ns 0.5X VD  0.5X 630 ns  A  B 2 0.5X 630 ns 0.5X VD −0.5X 630 ns −A −B 30.5X 630 ns 0.5X VD  0.5Y 630 ns −B  A 4 0.5X 630 ns 0.5X VD −0.5Y 630ns  B −A 5 0.5X 630 ns −0.5X  VD  0.5X 630 ns −A −B 6 0.5X 630 ns −0.5X VD −0.5X 630 ns  A  B 7 0.5X 630 ns −0.5X  VD  0.5Y 630 ns  B −A 8 0.5X630 ns −0.5X  VD −0.5Y 630 ns −B  A 9 −0.5X 630 ns 0.5X VD  0.5X 630 ns−A −B 10 −0.5X 630 ns 0.5X VD −0.5X 630 ns  A  B 11 −0.5X 630 ns 0.5X VD 0.5Y 630 ns  B −A 12 −0.5X 630 ns 0.5X VD −0.5Y 630 ns −B  A 13 −0.5X630 ns −0.5X  VD  0.5X 630 ns  A  B 14 −0.5X 630 ns −0.5X  VD −0.5X 630ns −A −B 15 −0.5X 630 ns −0.5X  VD  0.5Y 630 ns −B  A 16 −0.5X 630 ns−0.5X  VD −0.5Y 630 ns  B −A 17 −0.5Y 630 ns 0.5X VD  0.5X 630 ns  B −A18 −0.5Y 630 ns 0.5X VD −0.5X 630 ns −B  A 19 −0.5Y 630 ns 0.5X VD  0.5Y630 ns  A  B 20 −0.5Y 630 ns 0.5X VD −0.5Y 630 ns −A −B 21 −0.5Y 630 ns−0.5X  VD  0.5X 630 ns −B  A 22 −0.5Y 630 ns −0.5X  VD −0.5X 630 ns  B−A 23 −0.5Y 630 ns −0.5X  VD  0.5Y 630 ns −A −B 24 −0.5Y 630 ns −0.5X VD −0.5Y 630 ns  A  B 25 0.5Y 630 ns 0.5X VD  0.5X 630 ns −B  A 26 0.5Y630 ns 0.5X VD −0.5X 630 ns  B −A 27 0.5Y 630 ns 0.5X VD  0.5Y 630 ns −A−B 28 0.5Y 630 ns 0.5X VD −0.5Y 630 ns  A  B 29 0.5Y 630 ns −0.5X  VD 0.5X 630 ns  B −A 30 0.5Y 630 ns −0.5X  VD −0.5X 630 ns −B  A 31 0.5Y630 ns −0.5X  VD  0.5Y 630 ns  A  B 32 0.5Y 630 ns −0.5X  VD −0.5Y 630ns −A −B π/2-FID; 70 ns π/2 RF pulses; 4-step phase cycling, 16640echoes, including phase cycling; 8 images with different repetitiontimes logarithmically spaced between 10 μs and 25 μs; imaging time 10minutes. First First Detection Detection N pulse delay channel Rechannel Im SFR 1 0.5X 1000 ns  A  B SPI 2 −0.5X 1000 ns −A −B 3 0.5Y1000 ns −B  A 4 −0.5Y 1000 ns  B −A π-T-π/2-did; 35 ns π/2 and π RFpulses; 4-step phase cycling applied only for detection sequence, 7520acquisitions per T, including phase cycling; 8 T's denoted as VD in thetable below logarithmically spaced between 0.5 μs and 16 μs; T^(LF) _(R)= 25 μs; imaging time 10 minutes. First First Second Second DetectionDetection N pulse delay pulse delay channel Re channel Im IRSPI 1 X VD0.5X 1000 ns  A  B 2 X VD 0.5X 1000 ns −A −B 3 X VD 0.5X 1000 ns −B  A 4X VD 0.5X 1000 ns  B −A

TABLE 2 Parameters of pulse sequences Transmitted Band- Average Pulse RFpower width Power Sequence Pulse length (W) (MHz) (W) 2pESE (T_(2e)) 35ns, π/2 and π 39.6(π/2), 158.5(π) 8.7 0.56 SFR ESE (T_(1e)) 35 ns, π/2and π 39.6(π/2), 158.5(π) 8.7 0.4 IRESE (T_(1e)) 35 ns, π/2 and π39.6(π/2), 158.5(π) 8.7 0.33 SE (T_(1e)) 60 ns, all π/2 12.6 9.7 0.1

TABLE 3 Relaxation times and precision for 0% O₂ 1 mM sample Standarddeviation T_(2e) or T_(1e) T_(2e) or T_(1e) Pulse Sequence (μs) (μs)2pESE (T_(2e)) 5.15 0.19 SFR ESE (T_(1e)) 6.2 1.4 IRESE (T_(1e)) 5.90.29 SE (T_(1e)) 5.8 0.38 Non-imaging relaxation times: T_(2e) = 5.07 μs(2pESE); T_(1e) = 5.8 μs (IRESE); T_(1e) = 5.87 μs (SE)

TABLE 4 Relaxation times and precision for 9.3% O₂ 1 mM sample. Standarddeviation T_(2e) or T_(1e) T_(2e) or T_(1e) Pulse Sequence (μs) (μs)2pESE (T_(2e)) 1.25 0.07 SFR ESE (T_(1e)) N/A* N/A* IRESE(T_(1e)) 1.310.15 SE(T_(1e)) 1.35 0.23 *the SFR sequence was unable to produceprecise measurement due to T_(1e) considerably smaller than the minimumrepetition time that can be achieve in our system. Non-imagingrelaxation times: T_(2e) = 1.24 μs (2pESE); T_(1e) = 1.33 μs (IRESE);T_(1e) = 1.31 μs (SE).

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In some embodiments, a series of measurements are made, each using a setof three excitation pulses. In some embodiments, each set of threepulses includes a pulse sequence, wherein each pulse has a phaserelative to X-Y polar coordinates, wherein a unity magnitude pulse withzero phase shift is considered to be an 1.0 X pulse (also called simplyan X pulse); a unity magnitude pulse with 0.5 pi radian (90 degrees)phase shift is considered to be an 1.0 Y pulse (also called simply a Ypulse); a unity magnitude pulse with 1.0 pi radian (180 degrees) phaseshift is considered to be an −1.0 X pulse (also called simply a −Xpulse); a unity magnitude pulse with 1.5 pi radians (270 degrees) phaseshift is considered to be an −1.0 Y pulse (also called simply a −Ypulse). In like manner, pulses having half that magnitude and a 0-degreephase shift are called 0.5X pulses, those with a 90-degree phase shiftare called 0.5Y pulses, those with a 180-degree phase shift are called−0.5X pulses, and those with a 270-degree phase shift are called −0.5Ypulses. In some embodiments, the durations of each of the first, secondand third pulse is about 35 ns (about 9 cycles of 250 MHz RF), with aphase delay denoted (+X, +Y, −X, and −Y) and a magnitude of unity (1) orhalf (0.5). The variable delay between the first pulse and the secondpulse is denoted VD1 (also denoted as “t” in FIG. 1B), and the variabledelay between the second pulse and the third pulse is denoted VD2 (alsodenoted as “r” in FIG. 1B). In some embodiments, a series of sets ofpulses are successively generated such that sixteen sets of three pulsesare generated for each of one or more first delays VD1 and each of oneor more second delays VD2, as follows:

First First Second Second Third Set pulse delay pulse delay pulse 1 −XVD1 +.5X VD2 +Y 2 −X VD1 −.5X VD2 +Y 3 −X VD1 +.5X VD2 −Y 4 −X VD1 −.5XVD2 −Y 5 −X VD1 +.5Y VD2 +X 6 −X VD1 −.5Y VD2 +X 7 −X VD1 +.5Y VD2 −X 8−X VD1 −.5Y VD2 −X 9 −X VD1 +.5Y VD2 +Y 10 −X VD1 −.5Y VD2 +Y 11 −X VD1+.5Y VD2 −Y 12 −X VD1 −.5Y VD2 −Y 13 −X VD1 +.5X VD2 +X 14 −X VD1 −.5XVD2 +X 15 −X VD1 +.5X VD2 −X 16 −X VD1 −5X VD2 −X

In some embodiments, after the third pulse, a third variable delay (alsodenoted as “r” in FIG. 1A2) is inserted before a period of the responseRF signal is acquired, wherein in some embodiments, the response RF isacquired for a period of about 4 to 6 microseconds. In some embodiments,the magnitude, quadrature (or phase) and/or frequency of the response RFare measured, digitized and stored for later analysis and/or imagereconstruction.

In some embodiments, the present invention provides an apparatus forelectron paramagnetic resonance imaging (EPRI) of a volume of animaltissue in vivo. This apparatus includes a set of surface transmit coils;and a set of surface receive coils, wherein the set of surface transmitcoils generates an excitation magnetic field in the volume of animaltissue in response to an applied electrical signal, and the set ofsurface receive coils generates a sensed electrical signal in responseto a sensed magnetic field in the volume of animal tissue, and whereinthe set of transmit coils and the set of receive coils are orientedrelative to one another such that the sensed electrical signal haslittle or no component directly due to the excitation magnetic field,and wherein the set of surface receive coils is configured to detectelectron paramagnetic resonance signals in the volume of animal tissue;and a pulsed-RF driver circuit, operatively coupled to the set oftransmit coils, that drives a RF pulse set having a plurality ofsuccessive RF pulses, including a first pulse having a plurality ofcycles of RF, followed by a first delay and thereafter by a second pulsethat includes a plurality of cycles of RF that are shifted in phase (byabout either zero radians (0 degrees), ½ pi radians (90 degrees), piradians (180 degrees) or 3/2 pi radians (270 degrees)) relative to thefirst π-pulse, followed by a second delay and thereafter by a thirdpulse that includes a plurality of cycles of RF that are shifted inphase (by about either zero radians (0 degrees), ½ pi radians (90degrees), pi radians (180 degrees) or 3/2 pi radians (270 degrees))relative to the first pulse. In some embodiments, the animal tissue ishuman tissue in a living human.

Some embodiments further include a magnetic-field generator configuredto generate a substantially static magnetic field in the volume ofanimal tissue, which is generally orthogonal to the excitation magneticfield and to the sensed magnetic field in the volume of animal tissue,and wherein the excitation magnetic field is generally orthogonal to thesensed magnetic field in the volume of animal tissue; an RF receivercircuit operatively coupled to the set of surface receive coils toreceive the sensed electrical signal from the set of surface receivecoils and to generate a received electrical signal; a digital-signalprocessor (DSP) unit operatively coupled to the RF receiver circuit andconfigured to process the received electrical signal and to generateimage data; a storage unit operatively coupled to the DSP unit toreceive and store the image data; and a display unit operatively coupledto the storage unit to receive and display the image data.

In some embodiments, the present invention provides a method forelectron paramagnetic resonance oxygen imaging (EPROI) of a volume ofanimal tissue in vivo in an animal. This method includes placing (e.g.,by injecting, ingesting, inhaling, swabbing or the like) a reportermolecule in the animal; applying an RF pulse sequence that elicits a T₁spin-lattice relaxation response from the volume of tissue; andgenerating an EPRI image of in the animal using the T₁ response.

In some embodiments, the present invention provides a method forelectron paramagnetic resonance imaging (EPRI) of a volume of animaltissue in vivo in an animal. This method includes generating asubstantially static magnetic field in the volume of animal tissue;generating a excitation set that includes a plurality of RF-excitationmagnetic-field pulses including a first pulse that includes a pluralityof cycles of RF, followed by a first delay and then a second pulse thatincludes a plurality of cycles of RF that are shifted in phase (by abouteither zero radians (0 degrees), ½ pi radians (90 degrees), pi radians(180 degrees) or 3/2 pi radians (270 degrees)) relative to the cycles ofthe first pulse, followed by a second delay and thereafter a third pulsethat includes a plurality of cycles of RF that are shifted in phase (byabout either zero radians (0 degrees), ½ pi radians (90 degrees), piradians (180 degrees) or 3/2 pi radians (270 degrees)) relative to thefirst pulse in the direction generally orthogonal to the substantiallystatic magnetic field in the volume of animal tissue from a surface ofthe animal next to the volume of animal tissue. The method furtherincludes sensing an RF magnetic field in a direction generallyorthogonal to the substantially static magnetic field in the volume ofanimal tissue from a surface of the animal next to the volume of animaltissue, wherein the sensed RF magnetic field is in a direction generallyorthogonal to the pulsed excitation magnetic field; generating areceived electrical signal based on the sensed RF magnetic field;digitally signal processing the received electrical signal to generateimage data; storing the image data; and displaying the image data.

In some embodiments, the present invention provides an apparatus and acorresponding method for improved signal-to-noise (S/N) measurementsuseful for electron paramagnetic resonance imaging (EPRI), in situ andin vivo, using high-isolation transmit/receive surface coils andtemporally spaced pulses of RF energy (e.g., in some embodiments, afirst pulse of about 9 cycles of an about-250-MHz signal) having anamplitude sufficient to invert or rotate the magnetization prepared inthe temporally static magnetic fields by 180 degrees (a so calledpi-pulse (π pulse)) followed closely, but at varied times, by a secondradio-frequency pulse of about 9 cycles of substantially the samefrequency but having an amplitude half that of the initial pulse torotate the magnetization by, e.g., 90 degrees (a so called pi-over-twopulse (π/2 pulse)), to the horizontal plane where it evolves for a veryshort fixed time after which time a third radio-frequency pulsesufficient to rotate the magnetization by, e.g., 180 degrees, thatallows the formation of an echo (in some embodiments, the cycles of thesecond pulse and third pulse are obtained from the same signal source asthose of the first pulse but are phase shifted by respect to each otherby 0, 90, 180, or 270 degrees to reduce signal artifact), which, in someembodiments, provide improved microenvironmental images that arerepresentative of particular internal structures in the human body andspatially resolved images of tissue/cell protein signals responding toconditions (such as hypoxia) that show the temporal sequence of certainbiological processes, and, in some embodiments, that distinguishmalignant tissue from healthy tissue.

Embodiments within the scope of the present invention include acomputer-readable medium for carrying or having computer-executableinstructions or data structures stored thereon. Such computer-readablemedium may be any available medium, which is accessible by ageneral-purpose or special-purpose computer system. By way of example,and not limitation, such computer-readable medium can comprise physicalstorage medium such as RAM, ROM, EPROM, CD-ROM or other optical-diskstorage, magnetic-disk storage or other magnetic-storage devices, EEPROMor FLASH storage devices, or any other medium which can be used to carryor store desired program code means in the form of computer-executableinstructions, computer-readable instructions, or data structures andwhich may be accessed by a general-purpose or special-purpose computersystem. This physical storage medium may be fixed to the computer systemas in the case of a magnetic drive or removable as in the case of anEEPROM device (e.g., FLASH storage device). In some embodiments, thisphysical storage medium may be accessible and/or downloadable over theinternet.

In some embodiments, the present invention provides an apparatus forelectron paramagnetic resonance oxygen imaging (EPROI) of a volume ofanimal tissue in vivo. This apparatus includes: a set of surfacetransmit coils; and a set of surface receive coils, wherein the set ofsurface transmit coils generates an excitation magnetic field in thevolume of animal tissue in response to an applied electrical signal, andthe set of surface receive coils generates a sensed electrical signal inresponse to a sensed magnetic field in the volume of animal tissue, andwherein the set of transmit coils and the set of receive coils areoriented relative to one another such that the sensed electrical signalhas little or no component directly due to the excitation magneticfield, and wherein the set of surface receive coils is configured todetect electron paramagnetic resonance signals in the volume of animaltissue; a pulsed-RF driver circuit, operatively coupled to the set oftransmit coils, that drives a plurality of pulse sets, each pulse sethaving a plurality of successive transmitted pulses, including a firstpulse having a plurality of cycles of RF, followed by a first delay andthereafter by a second pulse that includes a plurality of cycles of RF,followed by a second delay and thereafter by a third pulse that includesa plurality of cycles of RF; and an RF receiver circuit operativelycoupled to the set of surface receive coils to receive the sensedelectrical signal from the set of surface receive coils and to generatea received electrical signal, wherein the transmitted pulses are ofmagnitudes and durations configured to measure T₁ spin-latticerelaxation in the volume of tissue.

In some embodiments of the apparatus, for different ones of theplurality of pulse sets, the second transmit pulses have RF cycles thatare shifted in phase by a selected different amount (by about either 0degrees, 90 degrees, 180 degrees or 270 degrees) relative to the firstpulse, and the third pulses have RF cycles that are shifted in phase bya selected different amount (by about either 0 degrees, 90 degrees, 180degrees or 270 degrees) relative to the first pulse.

In some embodiments of the apparatus, for each of the plurality of pulsesets:

-   -   the—first transmit pulse is a pi pulse having a magnitude and        duration selected to rotate an electron paramagnetic resonance        spin by pi radians;    -   the second transmit pulse is a pi/2 pulse having a magnitude and        duration selected to rotate an electron paramagnetic resonance        spin ½ pi radians; and    -   the third transmit pulse is a pi pulse having a magnitude and        duration selected to rotate an electron paramagnetic resonance        spin pi radians.

In some embodiments of the apparatus, for each of the plurality of pulsesets, the first, second and third transmit pulses form an inversionrecovery with electron-spin echo detection (IRESE) sequence.

In some embodiments of the apparatus, for each of the plurality of pulsesets, the first, second and third transmit pulses form an inversionrecovery with electron-spin echo detection (IRESE) sequence having aπ-pulse as the first pulse, a T delay as the first delay, a π/2-pulse asthe second pulse, a τ delay as the second delay, a π-pulse as the thirdpulse, a τ delay as the third delay, wherein the first, second and thirdpulses are each about 35 ns in duration, wherein the π/2 pulse rotates amagnetization π/2 radians and the π-pulses rotate a magnetization πradians, wherein τ=630 ns, wherein T has a value in a range of about 500ns to about 16,000 ns, and wherein the cycles of RF have a frequency ofabout 250 MHz.

In some embodiments of the apparatus, for each of the plurality of pulsesets, the first, second and third transmit pulses form an inversionrecovery with electron-spin echo detection (IRESE) sequence having aπ-pulse as the first pulse, a T delay as the first delay, a π/2-pulse asthe second pulse, a τ delay as the second delay, a π-pulse as the thirdpulse, a τ delay as the third delay, wherein the first, second and thirdpulses are each about 35 ns in duration, wherein the π/2 pulse rotates amagnetization π/2 radians and the π-pulses rotate a magnetization πradians, wherein τ=630 ns, wherein T has a value in a range of about 500ns to about 16,000 ns, wherein the cycles of RF have a frequency ofabout 250 MHz, wherein the plurality of pulse sets apply a sixteen-stepphase cycling, wherein about 7520 acquisitions are acquired per value ofT and include phase cycling, wherein eight T values that areapproximately logarithmically spaced between one-half microseconds (0.5μs) and sixteen microseconds (16 μs) are used, wherein T^(LF) _(R)=25μs, and wherein the phase cycling includes values selected from rows ofthe following table:

Detection Detection First First Second Second Third Third channel Rechannel Im pulse delay pulse delay pulse delay (real) (imaginary) X T0.5X 630 ns  X 630 ns  A  B X T 0.5X 630 ns −X 630 ns  A  B X T 0.5X 630ns  Y 630 ns −A −B X T 0.5X 630 ns −Y 630 ns −A −B X T −0.5X 630 ns  X630 ns −A −B X T −0.5X 630 ns −X 630 ns −A −B X T −0.5X 630 ns  Y 630 ns A  B X T −0.5X 630 ns −Y 630 ns  A  B X T 0.5Y 630 ns  X 630 ns  B −A XT 0.5Y 630 ns −X 630 ns  B −A X T 0.5Y 630 ns  Y 630 ns −B  A X T 0.5Y630 ns −Y 630 ns −B  A X T −0.5Y 630 ns  X 630 ns −B  A X T −0.5Y 630 ns−X 630 ns −B  A X T −0.5Y 630 ns  Y 630 ns  B −A X T −0.5Y 630 ns −Y 630ns  B  −A.

Some embodiments of the apparatus further include a magnetic-fieldgenerator configured to generate a substantially static magnetic fieldin the volume of animal tissue, which is generally orthogonal to theexcitation magnetic field and to the sensed magnetic field in the volumeof animal tissue, and wherein the excitation magnetic field is generallyorthogonal to the sensed magnetic field in the volume of animal tissue;a digital-signal processor (DSP) unit operatively coupled to the RFreceiver circuit and configured to process the received electricalsignal and to generate image data; a storage unit operatively coupled tothe DSP unit to receive and store the image data; and a display unitoperatively coupled to the storage unit to receive and display the imagedata.

In some embodiments, the present invention provides a method forelectron paramagnetic resonance imaging (EPRI) of a volume of animaltissue in vivo in an animal. This method includes generating asubstantially static magnetic field in the volume of animal tissue; andgenerating a plurality of pulse sets, wherein the generating of each oneof the plurality of pulse sets includes: generating a first RFexcitation magnetic field pulse having a plurality of RF cycles in afirst direction generally orthogonal to the substantially staticmagnetic field in the volume of animal tissue from a surface of theanimal next to the volume of animal tissue; delaying for a first delaytime; generating a second RF excitation magnetic field pulse having aplurality of RF in the first direction generally orthogonal to thesubstantially static magnetic field; delaying for a second delay time;generating a third RF excitation magnetic field pulse having a pluralityof RF in the first direction generally orthogonal to the substantiallystatic magnetic field; delaying for a third delay time; sensing an RFspin-relaxation signal; and generating a received electrical signalbased on the sensed RF signal; wherein the first, second and third RFexcitation magnetic field pulses are of magnitudes and durationsconfigured to measure T₁ spin-lattice relaxation in the volume oftissue. In some embodiments, the animal tissue is human tissue in aliving human.

In some embodiments of the method, for different ones of the pluralityof pulse sets, the second transmit pulses have RF cycles that areshifted in phase by a selected different amount (by about either 0degrees, 90 degrees, 180 degrees or 270 degrees) relative to the firstpulse, and the third pulses have RF cycles that are shifted in phase bya selected different amount (by about either 0 degrees, 90 degrees, 180degrees or 270 degrees) relative to the first pulse.

In some embodiments of the method, for each of the plurality of pulsesets: the first transmit pulse is a pi pulse having a magnitude andduration selected to rotate an electron paramagnetic resonance spin bypi radians; the second transmit pulse is a pi/2 pulse having a magnitudeand duration selected to rotate an electron paramagnetic resonance spin½ pi radians; and the third transmit pulse is a pi pulse having amagnitude and duration selected to rotate an electron paramagneticresonance spin pi radians.

In some embodiments of the method, for each of the plurality of pulsesets, the first, second and third transmit pulses form an inversionrecovery with electron-spin echo detection (IRESE) sequence.

In some embodiments of the method, for each of the plurality of pulsesets, the first, second and third transmit pulses form an inversionrecovery with electron-spin echo detection (IRESE) sequence having aπ-pulse as the first pulse, a T delay as the first delay, a π/2-pulse asthe second pulse, a τ delay as the second delay, a π-pulse as the thirdpulse, a τ delay as the third delay, wherein the first, second and thirdpulses are each about 35 ns in duration, wherein the π/2 pulse rotates amagnetization π/2 radians and the π-pulses rotate a magnetization πradians, wherein τ=630 ns, wherein T has a value in a range of about 500ns to about 16,000 ns, and wherein the cycles of RF have a frequency ofabout 250 MHz.

In some embodiments of the method, for each of the plurality of pulsesets, the first, second and third transmit pulses form an inversionrecovery with electron-spin echo detection (IRESE) sequence having aπ-pulse as the first pulse, a T delay as the first delay, a π/2-pulse asthe second pulse, a τ delay as the second delay, a π-pulse as the thirdpulse, a τ delay as the third delay, wherein the first, second and thirdpulses are each about 35 ns in duration, wherein the π/2 pulse rotates amagnetization π/2 radians and the π-pulses rotate a magnetization πradians, wherein τ=630 ns, wherein T has a value in a range of about 500ns to about 16,000 ns, wherein the cycles of RF have a frequency ofabout 250 MHz, wherein the plurality of pulse sets apply a sixteen-stepphase cycling, wherein about 7520 acquisitions are acquired per value ofT and include phase cycling, wherein eight T values that areapproximately logarithmically spaced between one-half microseconds (0.5μs) and sixteen microseconds (16 μs) are used, wherein T^(LF) _(R)=25μs, and wherein the phase cycling uses values selected according to thefollowing table:

Detection Detection First First Second Second Third Third channel Rechannel Im N pulse delay pulse delay pulse delay (real) (imaginary) 1 XT 0.5X 630 ns  X 630 ns  A  B 2 X T 0.5X 630 ns −X 630 ns  A  B 3 X T0.5X 630 ns  Y 630 ns −A −B 4 X T 0.5X 630 ns −Y 630 ns −A −B 5 X T−0.5X 630 ns  X 630 ns −A −B 6 X T −0.5X 630 ns −X 630 ns −A −B 7 X T−0.5X 630 ns  Y 630 ns  A  B 8 X T −0.5X 630 ns −Y 630 ns  A  B 9 X T0.5Y 630 ns  X 630 ns  B −A 10 X T 0.5Y 630 ns −X 630 ns  B −A 11 X T0.5Y 630 ns  Y 630 ns −B  A 12 X T 0.5Y 630 ns −Y 630 ns −B  A 13 X T−0.5Y 630 ns  X 630 ns −B  A 14 X T −0.5Y 630 ns −X 630 ns −B  A 15 X T−0.5Y 630 ns  Y 630 ns  B −A 16 X T −0.5Y 630 ns −Y 630 ns  B  −A.

Some embodiments of the method further include digitally signalprocessing the received electrical signal to generate image data;storing the image data; and displaying the image data.

In some embodiments, the present invention provides an apparatus forelectron paramagnetic resonance imaging (EPRI) of a volume of animaltissue in vivo in an animal. This apparatus includes means (as describedherein and equivalents thereto) for generating a substantially staticmagnetic field in the volume of animal tissue; and means for generatinga plurality of pulse sets, wherein the means for generating each one ofthe plurality of pulse sets includes: means for generating a first RFexcitation magnetic field pulse having a plurality of RF cycles in afirst direction generally orthogonal to the substantially staticmagnetic field in the volume of animal tissue from a surface of theanimal next to the volume of animal tissue; means for delaying for afirst delay time; means for generating a second RF excitation magneticfield pulse having a plurality of RF in the first direction generallyorthogonal to the substantially static magnetic field; means for sensingan RF spin-relaxation signal; and means for generating a receivedelectrical signal based on the sensed RF signal; wherein the first,second and third RF excitation magnetic field pulses are of magnitudesand durations configured to measure T₁ spin-lattice relaxation in thevolume of tissue.

In some embodiments of this apparatus, for different ones of theplurality of pulse sets, the second transmit pulses have RF cycles thatare shifted in phase by a selected different amount (by about either 0degrees, 90 degrees, 180 degrees or 270 degrees) relative to the firstpulse, and the third pulses have RF cycles that are shifted in phase bya selected different amount (by about either 0 degrees, 90 degrees, 180degrees or 270 degrees) relative to the first pulse. In some embodimentsof this apparatus, for each of the plurality of pulse sets: the firsttransmit pulse is a pi pulse having a magnitude and duration selected torotate an electron paramagnetic resonance spin by pi radians; the secondtransmit pulse is a pi/2 pulse having a magnitude and duration selectedto rotate an electron paramagnetic resonance spin ½ pi radians; and thethird transmit pulse is a pi pulse having a magnitude and durationselected to rotate an electron paramagnetic resonance spin pi radians.In some embodiments of the apparatus, for each of the plurality of pulsesets, the first, second and third transmit pulses form an inversionrecovery with electron-spin echo detection (IRESE) sequence.

In some embodiments of the apparatus, for each of the plurality of pulsesets: the first, second and third transmit pulses form an inversionrecovery with electron-spin echo detection (IRESE) sequence having aπ-pulse as the first pulse, a T delay as the first delay, a π/2-pulse asthe second pulse, a τ delay as the second delay, a π-pulse as the thirdpulse, a τ delay as the third delay, wherein the first, second and thirdpulses are each about 35 ns in duration, wherein the π/2 pulse rotates amagnetization π/2 radians and the π-pulses rotate a magnetization πradians, wherein τ=630 ns, wherein the cycles of RF have a frequency ofabout 250 MHz, wherein the plurality of pulse sets apply a sixteen-stepphase cycling, wherein about 7520 acquisitions are acquired per value ofT and include phase cycling, wherein eight T values are approximatelylogarithmically spaced between one-half microseconds (0.5 μs) andsixteen microseconds (16 μs) are used, wherein T^(LF) _(R)=25 μs, andwherein the phase cycling uses values selected from rows of thefollowing table:

Detection Detection First First Second Second Third Third channel Rechannel Im pulse delay pulse delay pulse delay (real) (imaginary) X T0.5X 630 ns  X 630 ns  A  B X T 0.5X 630 ns −X 630 ns  A  B X T 0.5X 630ns  Y 630 ns −A −B X T 0.5X 630 ns −Y 630 ns −A −B X T −0.5X 630 ns  X630 ns −A −B X T −0.5X 630 ns −X 630 ns −A −B X T −0.5X 630 ns  Y 630 ns A  B X T −0.5X 630 ns −Y 630 ns  A  B X T 0.5Y 630 ns  X 630 ns  B −A XT 0.5Y 630 ns −X 630 ns  B −A X T 0.5Y 630 ns  Y 630 ns −B  A X T 0.5Y630 ns −Y 630 ns −B  A X T −0.5Y 630 ns  X 630 ns −B  A X T −0.5Y 630 ns−X 630 ns −B  A X T −0.5Y 630 ns  Y 630 ns  B −A X T −0.5Y 630 ns −Y 630ns  B −A

Some embodiments of the apparatus further include a magnetic-fieldgenerator configured to generate a substantially static magnetic fieldin the volume of animal tissue, which is generally orthogonal to theexcitation magnetic field and to the sensed magnetic field in the volumeof animal tissue, and wherein the excitation magnetic field is generallyorthogonal to the sensed magnetic field in the volume of animal tissue;a digital-signal processor (DSP) unit operatively coupled to the RFreceiver circuit and configured to process the received electricalsignal and to generate image data; a storage unit operatively coupled tothe DSP unit to receive and store the image data; and a display unitoperatively coupled to the storage unit to receive and display the imagedata.

It is to be understood that the above description is intended to beillustrative, and not restrictive. Although numerous characteristics andadvantages of various embodiments as described herein have been setforth in the foregoing description, together with details of thestructure and function of various embodiments, many other embodimentsand changes to details will be apparent to those of skill in the artupon reviewing the above description. The scope of the invention should,therefore, be determined with reference to the appended claims, alongwith the full scope of equivalents to which such claims are entitled. Inthe appended claims, the terms “including” and “in which” are used asthe plain-English equivalents of the respective terms “comprising” and“wherein,” respectively. Moreover, the terms “first,” “second,” and“third,” etc., are used merely as labels, and are not intended to imposenumerical requirements on their objects.

What is claimed is:
 1. An apparatus for electron paramagnetic resonanceimaging (EPRI) of a volume of animal tissue in vivo in an animal, theapparatus comprising: a magnetic-field generator configured to generatea substantially static magnetic field in the volume of animal tissue; aset of transmit coils, wherein the set of surface transmit coilsgenerates an excitation magnetic field in the volume of animal tissue inresponse to an applied electrical signal; and a set of receive coils,wherein the set of surface receive coils generates a sensed electricalsignal in response to a sensed magnetic field in the volume of animaltissue, and wherein the set of surface receive coils is configured todetect electron paramagnetic resonance signals in the volume of animaltissue; a pulsed-RF driver circuit, operatively coupled to the set oftransmit coils, that drives a plurality of pulse sets, each pulse setincluding: a first pulse that includes a plurality of cycles of RF,followed by a first delay and then a second pulse that includes aplurality of cycles of RF that are shifted in phase (by about eitherzero radians (0 degrees), ½ pi radians (90 degrees), pi radians (180degrees) or 3/2 pi radians (270 degrees)) relative to the cycles of thefirst pulse, followed by a second delay and thereafter a third pulsethat includes a plurality of cycles of RF that are shifted in phase (byabout either zero radians (0 degrees), ½ pi radians (90 degrees), piradians (180 degrees) or 3/2 pi radians (270 degrees)) relative to thefirst pulse in the direction generally orthogonal to the substantiallystatic magnetic field in the volume of animal tissue from a surface ofthe animal next to the volume of animal tissue; an RF receiver circuitoperatively coupled to the set of surface receive coils to receive thesensed electrical signal from the set of surface receive coils and togenerate a received electrical signal, wherein the transmitted pulsesets are of magnitudes and durations configured to measure an electronT_(1e) spin-lattice relaxation in the volume of tissue in order tomeasure oxygen in the volume of tissue; and an image-generation unitoperatively coupled to the RF receiver circuit and configured to processthe received electrical signal and to generate image data.
 2. Theapparatus of claim 1, wherein for each of the plurality of pulse sets,the first, second and third transmit pulses form an inversion recoverywith electron-spin echo detection (IRESE) sequence.
 3. The apparatus ofclaim 1, further comprising: a digital-signal processor (DSP) unitoperatively coupled to the RF receiver circuit and configured to processthe received electrical signal and to generate image data; a storageunit operatively coupled to the DSP unit to receive and store the imagedata; and a display unit operatively coupled to the storage unit toreceive and display the image data.
 4. An apparatus for electronparamagnetic resonance oxygen imaging (EPROI) of a volume of animaltissue in vivo, the apparatus comprising: a magnetic-field generatorconfigured to generate a substantially static magnetic field in thevolume of animal tissue; a set of transmit coils, wherein the set ofsurface transmit coils generates an excitation magnetic field in thevolume of animal tissue in response to an applied electrical signal; anda set of receive coils, wherein the set of surface receive coilsgenerates a sensed electrical signal in response to a sensed magneticfield in the volume of animal tissue, and wherein the set of surfacereceive coils is configured to detect electron paramagnetic resonancesignals in the volume of animal tissue; a pulsed-RF driver circuit,operatively coupled to the set of transmit coils, that drives aplurality of pulse sets, wherein each pulse set has a plurality ofsuccessive transmitted pulses, including a first pulse having aplurality of cycles of RF, followed by a first delay and thereafter by asecond pulse that includes a plurality of cycles of RF; and an RFreceiver circuit operatively coupled to the set of surface receive coilsto receive the sensed electrical signal from the set of surface receivecoils and to generate a received electrical signal, wherein thetransmitted pulses are of magnitudes and durations configured to measurean electron T_(1e) spin-lattice relaxation in the volume of tissue inorder to measure oxygen in the volume of tissue.
 5. The apparatus ofclaim 4, wherein for different ones of the plurality of pulse sets: thesecond transmit pulses have RF cycles that are shifted in phase by aselected different amount (by about either 0 degrees, 90 degrees, 180degrees or 270 degrees for different ones of the plurality of pulsesets) relative to the first pulse.
 6. The apparatus of claim 4, whereineach pulse set includes the first pulse, followed by the first delay andthereafter by the second pulse, wherein the second pulse is followed bya second delay and thereafter by a third pulse that includes a pluralityof cycles of RF.
 7. The apparatus of claim 6, wherein for different onesof the plurality of pulse sets, the second transmit pulses have RFcycles that are shifted in phase by a selected different amount (byabout either 0 degrees, 90 degrees, 180 degrees or 270 degrees) relativeto the first pulse, and the third pulses have RF cycles that are shiftedin phase by a selected different amount (by about either 0 degrees, 90degrees, 180 degrees or 270 degrees) relative to the first pulse.
 8. Theapparatus of claim 6, wherein for each of the plurality of pulse sets,the first, second and third transmit pulses form an inversion recoverywith electron-spin echo detection (IRESE) sequence.
 9. The apparatus ofclaim 6, wherein for each of the plurality of pulse sets, the first,second and third transmit pulses form an inversion recovery withelectron-spin echo detection (IRESE) sequence having a π-pulse as thefirst pulse, a T delay as the first delay, a π/2-pulse as the secondpulse, a τ delay as the second delay, a π-pulse as the third pulse,wherein the first, second and third pulses are each about 35 ns induration, wherein the π/2 pulse rotates a magnetization π/2 radians andthe π-pulses rotate a magnetization π radians, wherein τ=630 ns, whereinT has a value in a range of about 500 ns to about 16,000 ns, and whereinthe cycles of RF have a frequency of about 250 MHz.
 10. The apparatus ofclaim 6, wherein for each of the plurality of pulse sets, the first,second and third transmit pulses form an inversion recovery withelectron-spin echo detection (IRESE) sequence having a π-pulse as thefirst pulse, a T delay as the first delay, a π/2-pulse as the secondpulse, a τ delay as the second delay, a π-pulse as the third pulse, a tdelay as a third delay, wherein the first, second and third pulses areeach about 35 ns in duration, wherein the π/2 pulse rotates amagnetization π/2 radians and the π-pulses rotate a magnetization πradians, wherein τ=630 ns, wherein T has a value in a range of about 500ns to about 16,000 ns, wherein the cycles of RF have a frequency ofabout 250 MHz, wherein the plurality of pulse sets apply a sixteen-stepphase cycling, wherein about 7520 acquisitions are acquired per value ofT and include phase cycling, wherein eight T values that areapproximately logarithmically spaced between one-half microseconds (0.5μs) and sixteen microseconds (16 μs) are used, wherein T^(LF) _(R)=25μs, and wherein the phase cycling includes values selected from rows ofthe following table: Detection Detection First First Second Second ThirdThird channel Re channel Im pulse delay pulse delay pulse delay (real)(imaginary) X T 0.5X 630 ns  X 630 ns  A  B X T 0.5X 630 ns −X 630 ns  A B X T 0.5X 630 ns  Y 630 ns −A −B X T 0.5X 630 ns −Y 630 ns −A −B X T−0.5X 630 ns  X 630 ns −A −B X T −0.5X 630 ns −X 630 ns −A −B X T −0.5X630 ns  Y 630 ns  A  B X T −0.5X 630 ns −Y 630 ns  A  B X T 0.5Y 630 ns X 630 ns  B −A X T 0.5Y 630 ns −X 630 ns  B −A X T 0.5Y 630 ns  Y 630ns −B  A X T 0.5Y 630 ns −Y 630 ns −B  A X T −0.5Y 630 ns  X 630 ns −B A X T −0.5Y 630 ns −X 630 ns −B  A X T −0.5Y 630 ns  Y 630 ns  B −A X T−0.5Y 630 ns −Y 630 ns  B  −A.


11. The apparatus of claim 6, further comprising: a magnetic-fieldgenerator configured to generate a substantially static magnetic fieldin the volume of animal tissue, which is generally orthogonal to theexcitation magnetic field and to the sensed magnetic field in the volumeof animal tissue, and wherein the excitation magnetic field is generallyorthogonal to the sensed magnetic field in the volume of animal tissue;a digital-signal processor (DSP) unit operatively coupled to the RFreceiver circuit and configured to process the received electricalsignal and to generate image data; a storage unit operatively coupled tothe DSP unit to receive and store the image data; and a display unitoperatively coupled to the storage unit to receive and display the imagedata.
 12. The apparatus of claim 5, further comprising: a digital-signalprocessor (DSP) unit operatively coupled to the RF receiver circuit andconfigured to process the received electrical signal and to generateimage data; and a display unit operatively coupled to receive anddisplay the image data.
 13. An apparatus for electron paramagneticresonance imaging (EPRI) of a volume of animal tissue in vivo in ananimal, the apparatus comprising: means for generating a substantiallystatic magnetic field in the volume of animal tissue; means forgenerating a plurality of pulse sets, wherein the means for generatingeach one of the plurality of pulse sets includes: means for generating afirst RF excitation magnetic field pulse having a plurality of RF cyclesin a first direction generally orthogonal to the substantially staticmagnetic field in the volume of animal tissue from a surface of theanimal next to the volume of animal tissue, means for delaying for afirst delay time after the first RF excitation magnetic field pulse,means for generating a second RF excitation magnetic field pulse havinga plurality of RF in the first direction generally orthogonal to thesubstantially static magnetic field, means for sensing an RFspin-relaxation signal, and means for generating a received electricalsignal based on the sensed RF signal; wherein the first and second RFexcitation magnetic field pulses are of magnitudes and durationsconfigured to measure T_(1e) spin-lattice relaxation response in thevolume of tissue; and means for generating an EPRI image of the in vivoanimal tissue using the T_(1e) response.
 14. The apparatus of claim 13,wherein for different ones of the plurality of pulse sets: the secondtransmit pulses have RF cycles that are shifted in phase by a selecteddifferent amount (by about either 0 degrees, 90 degrees, 180 degrees or270 degrees for different ones of the plurality of pulse sets) relativeto the first pulse.
 15. The apparatus of claim 13, further comprising:means for delaying for a second delay time after the second RFexcitation magnetic field pulse; means for generating a third RFexcitation magnetic field pulse having a plurality of RF in the firstdirection generally orthogonal to the substantially static magneticfield.
 16. The apparatus of claim 15, wherein for different ones of theplurality of pulse sets: the second transmit pulses have RF cycles thatare shifted in phase by a selected different amount (by about either 0degrees, 90 degrees, 180 degrees or 270 degrees for different ones ofthe plurality of pulse sets) relative to the first pulse, and the thirdtransmit pulses have RF cycles that are shifted in phase by a selecteddifferent amount (by about either 0 degrees, 90 degrees, 180 degrees or270 degrees) relative to the first pulse.
 17. The apparatus of claim 15,wherein for each of the plurality of pulse sets: the first transmitpulse is a pi pulse having a magnitude and duration selected to rotatean electron paramagnetic resonance spin by pi radians; the secondtransmit pulse is a pi/2 pulse having a magnitude and duration selectedto rotate an electron paramagnetic resonance spin ½ pi radians; and thethird transmit pulse is a pi pulse having a magnitude and durationselected to rotate an electron paramagnetic resonance spin pi radians.18. The apparatus of claim 15, wherein for each of the plurality ofpulse sets, the first, second and third transmit pulses form aninversion recovery with electron-spin echo detection (IRESE) sequence.19. The apparatus of claim 15, wherein for each of the plurality ofpulse sets, the first, second and third transmit pulses form aninversion recovery with electron-spin echo detection (IRESE) sequencehaving a π-pulse as the first pulse, a T delay as the first delay, aπ/2-pulse as the second pulse, a τ delay as the second delay, a π-pulseas the third pulse, a τ delay as a third delay, wherein the first,second and third pulses are each about 35 ns in duration, wherein theπ/2 pulse rotates a magnetization π/2 radians and the π-pulses rotate amagnetization π radians, wherein τ=630 ns, wherein T has a value in arange of about 500 ns to about 16,000 ns, wherein the cycles of RF havea frequency of about 250 MHz, wherein the plurality of pulse sets applya sixteen-step phase cycling, wherein about 7520 acquisitions areacquired per value of T and include phase cycling, wherein eight Tvalues are approximately logarithmically spaced between one-halfmicroseconds (0.5 μs) and sixteen microseconds (16 μs) are used, whereinT^(LF) _(R)=25 μs, and wherein the phase cycling uses values selectedfrom rows of the following table: Detection Detection First First SecondSecond Third Third channel Re channel Im pulse delay pulse delay pulsedelay (real) (imaginary) X T 0.5X 630 ns  X 630 ns  A  B X T 0.5X 630 ns−X 630 ns  A  B X T 0.5X 630 ns  Y 630 ns −A −B X T 0.5X 630 ns −Y 630ns −A −B X T −0.5X 630 ns  X 630 ns −A −B X T −0.5X 630 ns −X 630 ns −A−B X T −0.5X 630 ns  Y 630 ns  A  B X T −0.5X 630 ns −Y 630 ns  A  B X T0.5Y 630 ns  X 630 ns  B −A X T 0.5Y 630 ns −X 630 ns  B −A X T 0.5Y 630ns  Y 630 ns −B  A X T 0.5Y 630 ns −Y 630 ns −B  A X T −0.5Y 630 ns  X630 ns −B  A X T −0.5Y 630 ns −X 630 ns −B  A X T −0.5Y 630 ns  Y 630 ns B −A X T −0.5Y 630 ns −Y 630 ns  B  −A.


20. The apparatus of claim 13, wherein the means for generating an EPRIimage includes: digital-signal-processing means operatively coupled toprocess the received electrical signal and to generate image data;storage means operatively coupled to receive and store the image data;and display means operatively coupled to the storage means to receiveand display the image data.